Self-Assembly of Synthetic and Biological Polymeric Systems of ...

Grupo de Física de Coloides y Polímeros
Departamento de Física de la Materia Condensada
Facultad de Física
Tesis doctoral, 2012
Self-Assembly of Synthetic and Biological
Polymeric Systems of Technological Interest
Josué Elías Juárez Onofre

Víctor Mosquera Tallón, Catedrático, y Pablo Taboada Antelo, Profesor Titular, ambos del
Departamento de Física de la Materia Condensada de la Universidad de Santiago de
Compostela,
INFORMAN:
Que el trabajo de investigación titulado “Self-assembly of Synthetic and Biological
Polymeric Systems of Technological Interest”, presentado por Josué Elías Juárez Onofre ha sido
dirigido por ellos en el Grupo de Física de Coloides y Polímeros del Departamento de Física de
la Materia Condensada de la Universidad de Santiago de Compostela, y reúne los requisitos de
calidad y rigor científicos necesarios para optar al Grado de Doctor en Ciencia y Tecnología de
Materiales.
Para que así conste a los efectos oportunos.
Santiago de Compostela, 6 de febrero de 2012
Fdo. Víctor Mosquera Tallón Fdo. Pablo Taboada Antelo

Agradecimientos
A mi Familia
A mi Padre y Madre

Nuestra vida está dividida en diferentes períodos y en cada uno de ellos siempre se
encuentran personas que influyen de forma esencial en su devenir, regalándonos su amistad y
compartiendo gran parte de su tiempo y conocimiento.
Primero, y de la manera más atenta, agradezco a mis supervisores de este trabajo, Víctor
Mosquera Tallón y Pablo Taboada Antelo, por el conocimiento y soporte científico que me han
dado durante la etapa académica y dirigiendo esta tesis doctoral. También agradecerles el
haber hecho lo “imposible” para que pudiera iniciar (ellos recordarán lo difícil y cómico de la
situación) y desarrollar este Doctorado en Ciencia y Tecnología de Materiales en la Universidad
de Santiago de Compostela.
También me gustaría agradecer al profesor Stephen Yeates por el apoyo científico y paciencia
durante mi estancia en la Facultad de Química de la Universidad de Manchester. También
quiero agradecer al profesor Miguel Valdéz de la Universidad de Sonora por su apoyo técnico y
científico.
Por otra parte, también mi más sincero agradecimiento mis compañeros-amigos-colegas del
Grupo de Física de Coloides y Polímeros: Emilio Castro, Sonia Goy, Silvia Barbosa, Adriana
Cambón, Antonio Topete y Manuel Alatorre, por formar parte de este período, compartiendo
su tiempo y conocimiento.
Me siento afortunado en tener a otros muchos compañeros-amigos-colegas y, por eso,
aprovecho para agradecerles su amistad incondicional, y haciendo uso de la poca memoria que
tengo trataré de citarlos en orden cronológico: Felipe Areais, Ceila Fong, Tareixa Regueira, Joel
Pinto, Bea Ordoñez, “Mary Joe”, “Mallegue”, Agueda, “Cío”, Hassan, Irais Batista y José Ríos,
Alejandra Parra, Natalia Hassan (Wua yu Min), Elena Blanco, Paula y Miguel, Liliana, Fernando,
“Toyis” y pido disculpas porque seguro olvidé a alguien. A mis amigos de la residencia Cadarso.
Quiero agradecer también al equipo de microscopía electrónica: Miro, Raquel y Merche, por el
tiempo y paciencia que dedicaron en enseñarme a manejar los equipos de TEM, microscopía
confocal y SEM.
A TODOS, MUCHAS GRACIAS!!!
iii

v
List of papers
I. Self-assembly process of different poly(oxystyrene)-poly(oxyethylene) block copolymers:
spontaneous formation of vesicular structures and elongated micelles. Juárez, J.; Taboada,
P.; Valdez, M. A.; Mosquera, V. 2008, Langmuir, Vol. 24, pp. 7107-7116.
II. Existence of different structural intermediates on the fibrillation pathway of human serum
albumin. Juárez, J.; Taboada, P.; Mosquera, V. 2009, Biophysical Journal, Vol. 96, pp.
2353-2370.
III. Influence of electrostatic interactions on the fibrillation process of human serum albumin.
Juárez, J.; Taboada, P.; Mosquera, V. 2009, J. Phys. Chem. B, Vol. 113, pp. 10521-10529.
IV. Additional supra-self-assembly of human serum albumin under amyloid-like-forming
solution conditions. Juárez, J.; Taboada, P.; Goy-López, S.; Cambón, A.; Madec, M. B.;
Yeates, S. G.; Mosquera, V. 2009, J. Phys. Chem. B, Vol. 113, pp. 12391-12399.
V. Surface properties of monolayers of amphiphilic poly(ethylene oxide)-poly(styrene oxide)
block copolymers. Juárez, J.; Goy-López, S.; Cambón, A.; Valdez, M. A.; Taboada, P.;
Mosquera, V. 2010, J. Phys. Chem. Vol. 114, pp. 15703-15712.
VI. Obtention of metallic nanowires by protein biotemplating and their catalytic application.
Juárez, J.; Cambón, A.; Goy-López, S.; Topete, A.; Taboada, P.; Mosquera, V. 2010, J.
Phys. Chem. Lett. Vol. 1 pp. 2680-2687.
VII. One-dimensional magnetic nanowires obtained by protein fibril biotemplating. Juárez, J.;
Cambón, A.; Topete, A.; Taboada, P.; Mosquera, V. 2011, Chem. Eur. J. Vol. 17 pp. 7366-
7373.
VIII. Hydration effects on the fibrillation process of a globular protein: The case of human
serum albumin. Juárez, J.; Alatorre-Meda, M.; Cambón, A.; Topete, A.; Barbosa, S.;
Taboada, P.; Mosquera, V. 2012, Soft Matter. DOI:10.1039/C2SM06762E.

vii
Índice general
Agradecimientos ii
Lista de artículos iv
Resumen 1
Abstract 11
CHAPTER 1
Synthetic and natural polymers
1.1
1.2
1.3
1.4
1.2.1
1.2.2
1.2.3
1.3.1
1.3.2
1.3.3
Chapter 2
Methods
2.1
2.2
2.2.1
2.2.2
2.2.3
Introduction
Classification of polymers
Synthetic polymers
Biopolymers
Proteins
(Bio)polymer structure
Primary structure
Secondary structure
Tertiary structure
Molecular self-assembly
Introduction
Surface tension
Wilhelmy plate method
Monolayers and Langmuir-Blodgett films
Axisymetric drop shape (ADSA)
15
16
17
18
19
19
20
20
22
23
28
28
28
29
31

2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.3.1
2.3.2
2.4.1
2.4.2
2.9.1
2.11.1
2.11.2
2.11.3
2.11.4
2.11.5
Chapter 3
Light scattering
Static light scattering (SLS)
Dynamic light scattering (DLS)
Electron microscopy
Transmission electron microscopy (TEM)
Scanning electron microscopy (SEM)
Atomic force microscopy (AFM)
Fluorescence spectroscopy
Circular dichroism (CD)
Infrared spectroscopy
Rheology
Viscoelasticity
X-ray diffraction
Additional experimental techniques used in fibrillogenesis
studies
Block copolymers
3.1
3.2
3.3
UV-Vis spectroscopy
Polarized light optical microscopy
Emission spectroscopy
Superconducting quantum interference device (SQUID)
Magnetic resonance imaging (MRI)
Laboratory equipment
Introduction
Behaviour of block copolymers in solution
Mechanisms of morphologic control and thermodynamic
stability
32
36
40
44
44
46
47
48
52
54
58
59
64
72
72
75
77
78
80
82
91
93
95
viii

3.4
3.5
Chapter 4
Papers on block copolymers
4.1
4.2
4.3
4.3.1
Chapter 5
Proteins
5.1
5.2
5.3
5.1.1
5.1.2
5.1.3
5.1.4
5.2.1
5.2.2
5.2.3
5.2.4
5.3.1
Block copolymers of polyoxyalkylenes in aqueous solution
E12S10, E10S10E10 and E137S18E137 block copolymers
Self-Assembly process of different poly(oxystyrene)-
poly(oxyethylene) block copolymers: spontaneous
formation of vesicular structures and elongated micelles.
Surface properties of monolayers of amphiphilic
poly(ethylene oxide)-poly(styrene oxide) block copolymers.
Appended papers
Relevant aspects
Protein structure
Primary structure of the proteins
Secondary structure of the proteins
Tertiary structure of the proteins
Quaternary structure of the proteins
Protein aggregation
Unfolded state
Intermediate and transition state assembles
Native state
Energy landscape: protein aggregation
Amyloid fibrils
Amyloid fibrils hallmarks
96
97
103
113
123
124
127
128
129
131
132
132
133
133
134
135
135
135
ix

5.4
5.5
5.3.2
5.5.1
Chapter 6
Kinetic fibril formation in vitro
Human serum albumin
Papers on amyloid fibrils
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
Amyloid fibrils as biomaterials
Hen egg-white lysozyme
Existence of different structural intermediates on the
fibrillation pathway of human serum albumin
Influence of electrostatic interactions on the fibrillation
process of human serum albumin
Additional supra-self-assembly of human serum albumin
under amyloid-like-forming solution conditions
Hydration effects on the fibrillation process of globular
protein: The case of human serum albumin
Obtention of metallic nanowires by protein biotemplating
and their catalytic application
One-dimensional magnetic nanowires obtained by protein
fibril biotemplating
Relevant aspects on the HSA fibril formation
Relevant aspects on the gold and magnetic nanowires
Conclusions 219
References 223
138
139
141
142
147
165
175
185
199
207
215
216
x

Resumen
Desde tiempos ancestrales, las moléculas poliméricas, también conocidas como macromoléculas o
moléculas gigantes, han representado un material útil para el desarrollo de la humanidad.
Actualmente, es difícil imaginar el desarrollo de una sociedad organizada sin el uso de materiales
poliméricos. Por ejemplo, desde hace algunas décadas hasta nuestros días, estos materiales
poliméricos son utilizados para satisfacer diferentes necesidades de gran importancia como son la
construcción de refugios, viviendas (casas, residencias, etc.) y vestimenta; dependiendo del uso
que se requiera, se emplearán materiales poliméricos sintéticos (tales como el poliéster,
polietileno, polipropileno, teflón, resinas, entre otros) y/o naturales, también conocidos como
biopolímeros (madera, pieles, gomas y resinas naturales, algodón seda, lana). Por otra parte,
diferentes macromoléculas desempeñan una función de gran importancia biológica.
Biomacromoléculas como los ácidos ribonucleicos (ADN y ARN), proteínas y polisacáridos son de
vital importancia en distintos procesos bioquímicos para el crecimiento y desarrollo de un
organismo vivo; además, proteínas y polisacáridos son utilizados por los organismos vivos como
soportes estructurales de células, tejidos y órganos.
El uso de los materiales poliméricos no sólo está limitado a los usos descritos anteriormente. Hoy
en día una gran variedad de materiales poliméricos, tanto sintéticos como naturales, están siendo
utilizados en diferentes áreas tecnológicas como la ciencia de materiales, medicina clínica o
industria farmacéutica, entre otras. Además, los procesos de síntesis química, hacen posible
diseñar una macromolécula con propiedades fisicoquímicas deseadas. Con este fin, diversos
grupos de investigación han sintetizado diferentes tipos de copolímeros con carácter anfifílico. El
diseño de la arquitectura molecular del copolímero puede ser tan sencillo como un polímero
lineal, o tan complejo como un copolímero de injerto o tipo estrella; la arquitectura molecular
puede controlarse por medio de procedimientos de síntesis química.
Nuestro trabajo de investigación se divide en dos etapas: la primera parte se enfocó a la
caracterización estructural y de las propiedades físico-químicas de tres copolímeros de bloque con
potencial interés para ser empleados como sistemas de liberación farmacológica; mientras que en
la segunda parte se ha realizado un estudio del proceso de autoensamblaje en la forma de fibras
1

amiloides de la proteína albúmina de suero humano (HSA), su caracterización estructural y de sus
propiedades físico-químicas, y su uso potencial como biomateriales.
Debido a que parte de este trabajo consiste en la caracterización de tres copolímeros de bloque,
describiremos brevemente este tipo de macromoléculas. Para un copolímero, el “esqueleto” de la
cadena macromolecular está constituido por más de un monómero o unidad de repetición. Por lo
tanto, la estructura molecular de un copolímero de bloque consiste en la unión química de dos o
más macromoléculas de homopolímeros diferentes. De acuerdo al número de bloques (unidades
homopoliméricas) estos polímeros se denominan copolímeros dibloque, tribloque o multibloque.
Generalmente, estos homopolímeros son de diferente naturaleza (hidrófilo o hidrófobo)
proporcionando a la macromolécula un carácter anfifílico.
Bajo ciertas condiciones, los copolímeros pueden adoptar una configuración tipo gusano o
espagueti, mientras que para otras, el polímero puede auto-asociarse hasta formar
nanoestructuras regulares. Por ejemplo, si un copolímero de bloque se encuentra en disolución
acuosa, en donde el agua es un solvente selectivo para uno de los bloques, los segmentos
hidrófobos de las macromoléculas tienden a auto-asociarse, formando estructuras específicas,
evitando el contacto con las moléculas del disolvente. La auto-asociación origina un amplio rango
de comportamientos de fases, en los que se incluye la formación de micelas, nanovarillas,
vesículas o grandes agregados. Estas estructuras poliméricas presentan, en particular, un especial
interés en el área biomédica. Así, las estructuras micelares y vesiculares pueden ser utilizados
como sistemas de liberación de fármacos y proteínas, así como en el desarrollo de nanoreactores y
de biosensores.
Los copolímeros de bloque de oxialquilenos están constituidos, generalmente, por el bloque
hidrófilo polióxido de etileno (OCH2CH2 es la unidad de óxido de etileno, EO) y por un segundo
bloque hidrófobo que puede ser: un bloque de polióxido de propileno (PO), un bloque de
polióxido de estireno (SO) u otro tipo de bloque hidrófobo. En medio acuoso, la estructura de los
agregados que forman estos copolímeros de bloque está gobernada por el balance de las fuerzas
de interacción entre los bloques moleculares que constituyen el esqueleto del copolímero con las
moléculas de agua, y por las interacciones intermoleculares entre las cadenas del copolímero (ya
2

que las cadenas hidrófilas se expanden evitando las interacciones entre segmentos que no sean
favorables, mientras que las hidrófobas se contraen para minimizar el contacto con las moléculas
del disolvente). Este tipo de interacciones pueden modificarse por medio de factores físicos y, de
esta manera, se puede controlar la arquitectura de los agregados. Los factores físicos que se varían
con más frecuencia son: el peso molecular, la longitud relativa de los bloques, la estructura de los
bloques hidrófobos, las propiedades en disolución, etc. De esta forma, se ha observado que en
disoluciones acuosas diluidas estos copolímeros de bloque anfifílicos se asocian para formar
micelas esféricas. Al aumentar la concentración del copolímero se obtienen mesofases liotrópicas
cristalinas (geles), en donde las micelas se encuentran altamente empaquetadas en una estructura
cúbica centrada en las caras (fcc) o centrada en el cuerpo (bcc).
En el presente trabajo, se estudió el proceso de micelización, gelificación y la estructura de los
agregados de tres copolímeros de poli(óxido de etileno)-poli(óxido de estireno): EO12SO10,
EO10SO10EO10, y EO137SO18EO137, donde los subíndices indican la longitud de cada bloque. De estos
polímeros, dos tienen la misma longitud del bloque de poli(óxido de estireno) (EO12SO10 y
EO10SO10EO10,), mientras que la longitud del bloque EO para el tercer copolímero es mucho más
largo (EO137SO18EO137). Es importante mencionar que estos copolímeros de bloque fueron
sintetizados en colaboración con los Profs. David Attwood y Colin Booth de la en la School of
Pharmacy and Pharmaceutical Sciences y en la School of Chemistry of the University of
Manchester, respectivamente. La caracterización de estos copolímeros de bloque se realizó en dos
fases: La primera parte consistió en el análisis de las propiedades estructurales y físico-químicas de
los copolímeros en el seno de disoluciones acuosas, mediante diversas técnicas experimentales
tales como: medidas de tensión superficial, medidas de dispersión de luz láser (DLS y SLS),
microscopía óptica de luz polarizada (POM), microscopía de transmisión electrónica (TEM) y
microscopía electrónica de barrido (SEM). Por medio de las medidas experimentales para los
copolímeros EO12SO10, EO10SO10EO10, y EO137SO18EO137, combinado con modelos teóricos (según los
datos experimentales obtenidos) ya publicados, se determinaron la concentración micelar crítica,
así como propiedades micelares (tamaño de las micelas y número de agregación) y vesiculares
(para el caso del copolímero EO12SO10); imágenes de los agregados estructurados y el
comportamiento reológico. En este trabajo, en particular se observó por primera vez la posibilidad
de que los copolímeros de poli(óxido de etileno)-poli(óxido de estireno) pueden formar
estructuras vesiculares, sin necesidad de aportes externos de energía. Estas estructuras
3

vesiculares son el resultado de la auto-asociación del copolímero EO12SO10 en disolución acuosa, y
se encuentran en coexistencia con estructuras micelares esféricas. Este resultado se confirmó por
dispersión de luz láser, TEM y cryo- SEM. El tamaño de las vesículas varía entre 60 y 500 nm. Para
los copolímeros EO10SO10EO10 y EO137SO18EO137 se observaron estructuras en la forma de micelas
alargadas y esféricas, respectivamente. Comparando el alto número de agregación obtenido para
las micelas del copolímero EO10SO10EO10 por dispersión de luz láser con el número máximo de
agregación teórico que correspondería a una micela esférica, se puede concluir que la geometría
micelar no se corresponde con una geometría esférica, pero sí con una geometría más alargada.
Este resultado es confirmado por medio de imágenes obtenidas por TEM. La prueba de inversión
de tubo se utilizó para definir las fronteras que dividen la fase de un gel blando con la fase de un
gel duro de las disoluciones del copolímero EO137SO18EO137. De acuerdo con los resultados
obtenidos por reología, los copolímeros EO12SO10 y EO10SO10EO10 no forman geles en el rango de
concentraciones estudiados en este trabajo.
Por otra parte, la capacidad de adsorción y de auto-organización molecular en una interface de un
copolímero de bloque para formar nanoestructuras bien definidas en dos dimensiones permiten
que estos materiales poliméricos puedan utilizarse como estabilizantes coloidales, o como agentes
recubridores y estabilizantes de espumas y emulsiones.
Así, un amplio rango de estudios han analizado el comportamiento interfacial de copolímeros de
poli(óxido de etileno)-poli(óxido de propileno)-poli(óxido de etileno) (EO-PO-EO) ó “Pluronics”,
copolímeros de estrella de EO-PO-EO, también conocidos como “Tetronics” y de los copolímeros
poli(óxido de butileno)-poli(óxido de etileno) (BO-EO). Sin embargo, pocos estudios existen sobre
la caracterización en la interfase de los copolímeros de poli(óxido de etileno)-poli(óxido de
estireno). El análisis de sus propiedades de adsorción es interesante ya que puede proporcionar
importante información sobre su estabilidad intefacial y posterior agregación. Así, en el presente
trabajo, se estudiaron las propiedades de adsorción en la interface aire-agua y en la interface
cloroformo-agua de los copolímeros EO12SO10, EO10SO10EO10 y EO137SO18EO137. El trabajo de
investigación se desarrolló mediante las técnicas experimentales: tensiómetro de gota ,
monocapas o películas de Langmuir-Blodgett y microscopía de fuerza atómica (AFM). Las cinéticas
de adsorción de las disoluciones copoliméricas, en ambas interfaces, se determinaron por la
técnica de gota suspendida. Los resultados de tensión superficial muestran que la adsorción en la
4

interface aire-agua de los copolímeros estudiados disminuye conforme se incrementa la
hidrofobicidad del copolímero. Los datos obtenidos mediante reología interfacial de la película
copolimérica adsorbida en la interface aire-agua muestran que la capa adsorbida en la interface se
comporta como un sólido en todo el rango de frecuencias analizadas, mientras que la capa
polimérica adsorbida en la interface cloroformo-agua se comporta como un fluido viscoso para el
mismo rango de frecuencias. Las isotermas de compresión, presión superficial vs área ( vs A), y
las monocapas de Langmuir-Blodgett se obtuvieron con una balanza de Langmuir-Blodgett. Las
diferencias en las isotermas de Langmuir obtenidas para los tres copolímeros de bloque tienen su
origen en la diferente relación entre los bloques hidrófobo/hidrófilo. Bajo esta consideración, el
copolímero EO137SO18EO137 muestra una isoterma de adsorción con cuatro estados característicos,
mientras que en las isotermas de adsorción de los otros dos copolímeros (EO12SO10 y
EO10SO10EO10), sólo se observan dos regiones. Las monocapas de Langmuir-Blodgett se obtuvieron
a dos diferentes valores de presión superfcial (5 y 11 mN/m), y sus imágenes topográficas fueron
analizadas por AFM. Para ambas presiones, las imágenes topográficas revelan la existencia de
micelas circulares en la superficie de la película. Sin embargo, el tamaño de las micelas disminuye
con el aumento de la presión interfacial y, a su vez se observa un incremento del espesor de la
monocapa.
En la segunda etapa de nuestro trabajo, se estudiaron los procesos de ensamblaje molecular en la
forma de fibras en disolución acuosa de la proteína seroalbúmina humana (HSA, sus siglas en
inglés), bajo diferentes condiciones de temperatura, pH y fuerza iónica; y la posible aplicación de
estas nanoestructuras en diversas aplicaciones como catalizadores o elementos de contraste de
imagen.
In vivo, la agregación incontrolada de ciertas proteínas está relacionada directamente con
diferentes dolencias que afectan la salud humana. Uno de los productos de mayor interés
originados por la inadecuada agregación proteica son las fibras amiloides. Estas estructuras
fibrilares se han detectado en con más de 20 enfermedades (neurodegenerativas o no) tales como
el Alzheimer, Huntington, el mal de Parkinson, o la diabetes tipo II, entre otras. La capacidad de
fibrilación es independiente de la estructura nativa de la proteína, no obstante, la estructura
proteica primaria aparece como un factor importante en las primeras fases del proceso de
formación de las fibras amiloides, en su cinética de formación y en su estabilidad. La fibrilación se
5

origina debido a condiciones ambientales en las que se promueve la desnaturalización parcial o
completa de la estructura nativa de la proteína, por lo que para una proteína o cadena
polipeptídica, la formación de la fibra amiloide representa una estructura estable alternativa a la
estructura nativa.
Las fibras tipo amiloide presentan características termodinámicas y estructurales bien definidas
Estas fibras se caracterizan, en general, por ser lineales, rígidas y sin ramificaciones, con una
anchura que varía entre los 5-12 nm, tal como se ha observado por microscopía de transmisión
electrónica; además, tienen la propiedad de enlazarse a marcadores orgánicos específicos, como
Congo Red y Tioflavina T. Muestran también un patrón común de difracción de rayos-X
característico para una estructura -cruzada. Como se mencionó anteriormente, las fibras de
amiloide son responsables de distintas enfermedades como el mal de Parkinson, deficiencia de 1-
tripsina, Alzheimer y encefalopatías espongiformes. El origen de estas enfermedades está asociado
a la conversión de proteínas monoméricas a agregados proteicos insolubles, los cuales se
depositan en los tejidos/órganos del cuerpo.
La proteína plasmática más abundante del sistema circulatorio es la albúmina (representa
aproximadamente del 52 al 60 % de las proteínas plasmáticas), cuyas funciones son la regulación
de la presión osmótica sanguínea y en el transporte y almacenamiento de moléculas endógenas y
exógenas (hormonas, iones, ácidos grasos, drogas). A nivel industrial, la albúmina tiene un rol
importante en la tecnología médica, de alimentos, fármacos y en el desarrollo de biosensores
debido a sus fuertes propiedades de adsorción. La HSA es una proteína globular formada por una
sola cadena polipeptídica. La estructura de esta proteína está constituida por 585 residuos de
aminoácidos, con un peso molecular de aproximadamente 66,500 g/mol. La estructura secundaria
de la HSA consta principalmente de hélices (60 %); además presenta 17 puentes disulfuro, los
que le confiere una gran estabilidad estructural. La estructura globular de la HSA está compuesta
por tres dominios principales y seis subdominios unidos por puentes disulfuro. Debido a su
importancia fisiológica como proteína de transporte y reguladora de la presión osmótica, y a su
facilidad de agregación in vitro, la HSA se puede considerar como un modelo adecuado para
realizar estudios sobre los mecanismos de agregación proteica, con el fin de obtener una
aproximación y un mejor entendimiento de los mecanismos de agregación proteica asociada a la
amiloidogénesis. Ya que la estructura nativa de la HSA carece de cualquier predisposición a formar
6

fibras amiloides, la agregación de la albúmina sérica se puede promover cambiando las
condiciones de disolución (tales como de pH, altas temperaturas y por medio de agentes químicos
desnaturalizantes), favoreciendo la desestabilización de la estructura nativa de la HSA.
Así, en este trabajo se analizó la predisposición a la fibrilación de la HSA bajo diferentes
condiciones de disolución (pH, temperatura, fuerza iónica, composición del disolvente), así como
las propiedades físicas y estructurales de las fibras amiloidales resultantes. Las cinéticas de
agregación, los cambios conformacionales durante la auto-asociación de los monómeros de
albúmina y las estructuras de las diferentes estructuras intermedias en la ruta de fibrilación se
determinaron por fluorescencia de Tioflavina T (ThT) y absorbencia del Congo red; dicroísmo
circular (CD); fluorescencia intrínseca de la HSA; espectroscopía de infra-rojo (FTIR); difracción de
rayos-X; TEM; SEM y AFM. El proceso de la fibrilación de la HSA se prolonga durante varios días de
incubación, y se lleva a cabo sin la presencia de una fase de demora; excepto cuando las
disoluciones de albúmina se incubaron a pH ácido, temperatura ambiente y en ausencia de
electrólito. La ausencia de la fase de demora se debe al hecho de que la agregación inicial está
dirigida por el mecanismo denominado “cuesta abajo”, que no requiere de una alta organización
estructural ni de la formación de núcleos proteicos inestables. El proceso de fibrilación de la HSA
está acompañada por un incremento progresivo de la estructura secundaria hoja-β y estructuras
ordenadas al azar; esta transición se lleva a cabo a expensas de la estructura α-hélice, como se
observó mediante espectroscopía de fluorescencia de ThT, de CD y de FTIR. De acuerdo a las
distintas condiciones de incubación, la estructura nativa de la proteína se altera de distinta
manera/grado, implicando una variación en el contenido de las diferentes estructuras secundarias;
estas variaciones implican la aparición de diversos agregados estructurales que se forman en el
proceso de agregación fibrilar. Estos agregados pueden tomar la forma de: clústeres oligoméricos
globulares, estructuras rígidas esféricas y agregados en forma de anillos. La formación fibrilar
puede tener lugar a través de la asociación de los intermedios oligoméricos mencionados,
conduciendo a la formación de protofibras y, posteriormente, fibras. Estas fibras resultantes son
alargadas, con ondulaciones. La longitud de las fibras obtenidas depende de las condiciones de
incubación. Probablemente las hebras-β que componen la estructura de hoja-β en las fibras de
HSA se encuentran en un arreglo antiparalelo. La aparición de fibras amiloides maduras con
estructuras morfológicas más complejas tiene lugar a largos periodos de incubación (fibras largas y
rígidas, estructuras planas y alargadas, etc.). La tendencia de los oligómeros proteicos a alinearse
7

con las fibras vecinas más cercanas puede resultar en el crecimiento y maduración de las fibras
rígidas. Además, estas fibras pueden asociarse en otras estructuras supramoleculares tales como
esferulitas e incluso geles fibrilares. La presencia de esferulitas de fibras amiloides se confirmó por
POM, microscopía confocal y TEM. La HSA tiene la capacidad de formar geles fibrilares
entrecruzados, los cuales se forman por medio de interacciones intermoleculares no específicas a
valores de pH que se encuentran lejos del punto isoeléctrico de la HSA, cuando se sobrepasa un
umbral de concentración de proteínas y/o de fuerza iónica (punto de gelificación), tal como se
observó por reología. Por otra parte, cuando la HSA se encuentra cerca de su punto isoeléctrico, el
proceso de agregación proteica es tan rápido, que no permite una organización estructural
substancial para permitir la formación de estructuras ordenadas, formando así un gel de tipo
particulado.
A pesar de la importancia de las fibras amiloides en el área clínica, actualmente existen otras áreas
científicas que prestan especial interés en el estudio de las propiedades estructurales y de
agregación fibrilar, en la búsqueda de nuevos materiales y posibles aplicaciones en campos de
ingeniería y nanotecnología. El desarrollo de estructuras a escala mesoscópica es de gran
importancia en el desarrollo de dispositivos opto-electrónicos, como biosensores, materiales
biocompatibles, etc. Usualmente, los materiales nanoestructurados se obtienen a través de dos
estrategias: La primera, llamada “top-down”, consiste en la reducción de tamaño de un material
más grande; sin embargo, mediante esta estrategia se obtienen materiales en escala micrométrica
debido a que la reducción del tamaño sólo se puede realizar hasta donde las propiedades del
tamaño del material lo permita. En la segunda estrategia, “bottom-up”, se utilizan moléculas
pequeñas, como las proteínas, péptidos, fosfolípidos y ácidos nucleicos (ADN y RNA) como
elementos constituyentes, que bajo condiciones controladas, permiten obtener estructuras bien
ordenadas gracias a las propiedades de auto-ensamblaje que poseen este tipo de biomoléculas.
Las propiedades de auto-asociación y ensamblaje que tienen estos biomateriales permiten, bajo
condiciones controladas, obtener complejos nanoestructurados definidos. Las proteínas son una
alternativa muy atractiva para la construcción de estas nanoestructuras, ya que además de tener
un tamaño físico adecuado una gran estabilidad (comparado con los estructutras basadasen ácidos
nucleicos y fosfolípidos), y unas excelentes propiedades de adsorción a sustratos como vidrio,
óxido de silicio, u oro; por otra parte las proteínas presentan una serie de funciones que pueden
8

ser acopladas, por ejemplo, a circuitos electrónicos durante la construcción de nanodispositivos
gracias a su alta flexibilidad, aún cuando estos materiales son no conductores, pueden recubrirse
con metales conductores.
En la parte final de nuestro trabajo hemos analizado el potencial de las fibras amiloides como
nuevos nanomateriales con potenciales aplicaciones en distintos campos como la catálisis o la
biomedicina Bajo este contexto, en perime lugar se obtuvieron nanocables de oro por medio de
un proceso de crecimiento de “semillas” de oro utilizando fibras de la proteína lisozima como
bioplantillas. Se decidió emplear lisozima pues sus fiobras han sido muy estudiadas y7 presnetan
una geomtería recta sin ramificaciones, muy adecuada para el objetivo de la construcción de un
nanocable metálico. El grado de metalización de la fibra se controló por la adicción secuencial de
sal de oro hasta alcanzar un recubrimiento completo de la fibra. Los híbridos orgánico-metálicos
obtenidos se utilizaron como catalizadores en la reducción del p-nitrofenol a p-aminofenol.
Por otra parte, se obtuvieron nanocables magnéticos utilizando como bioplantillas las fibras de
lisozima y de HSA. La obtención de estos materiales híbridos se realizó por medio de una reacción
de precipitación in situ de la mezcla de sales de hierro Fe +2 :Fe +3 en una relación 1:2, en presencia
de NH4OH, siendo el producto de esta reacción nanopartículas de magnetita. Las propiedades
magnéticas de los híbridos se determinaron mediante SQUID (por sus siglas en inglés:
superconducting quantum interference device), y se compararon con las propiedades magnéticas
de nanopartículas libres de magnetita, observándose una mejora sustancial en sus propiedades
magnéticas. Finalmente, la posible utilidad de estos materiales biohíbridos como agentes de
contraste se corroboró mediante imágenes de resonancia magnética nuclear a diferentes
proporciones de dopado de la fibra proteica.
9

Abstract
In the present work, we investigated the micellization, gelation and the structure of aggregates of
three poly(ethylene oxide)-poly(styrene oxide)-poly(ethylene oxide) block copolymers (EO12SO10,
EO10SO10E10 and EO137SO18EO137, where E represents the oxyethylene unit, S the oxystyrene unit,
and the subscripts correspond to the number of monomeric unit constituting the polymeric chain)
in solution. We also investigated the adsorption process and the surface properties of these block
copolymers at the air-water (a/w) and chloroform-water (c/w) interfaces. Since these copolymers
possess a more hydrophobic middle block –the styrene oxide unit, they should gives rise to more
efficient drug delivery systems, in which geometry might play a key role for drug solubilisation and
cell uptake. For these reasons, a detailed characterization of the physicochemical properties of
these copolymers is required.
In this work, the spontaneous formation of vesicles by the block copolymer EO12SO10 without the
need of external energy input has been observed In aqueous bulk solution. This is the first time
that vesicle formation for styrene oxide based-block copolymers is reported. These vesicular
structures are in coexistence with spherical micelles, as confirmed by dynamic light scattering
(DLS), polarized optical microscopy and transmission and cryo-scanning electron microscopies.
Meanwhile, for block copolymers EO10SO10E10 and EO137SO18EO137 only one species is found in
solution, assigned to elongated and spherical micelles, respectively. The adsorption kinetics of
these block copolymers at both (a/w) and (c/w) interfaces were determined by the pendant drop
method. Measurements of their interfacial rheological behaviour showed that the adsorption
layer at the (a/w) interface manifests solid-like properties in the whole accessible frequency range,
whereas a viscous fluid-like behaviour is displayed at the (c/w) interface. The adsorption isotherm
for the block copolymer EO137SO18EO137 displays the four classical regions (pancake, mushroom,
brush, and condensed states), whereas for EO12SO10 and EO10SO10E10 copolymers only two regions
were observed. Probably, these observed differences in the monolayer isotherms arose from their
different hydrophobic/hydrophilic block ratios and block lengths.
On the other hand, the fibrillation propensity of the protein human serum albumin (HSA) was
analysed under different solution conditions (varying pH, temperature, ionic strength and solvent
11

composition). Fibrillation is an important self-assembly mechanism of proteins which allows them
to achieve an energy state even lower than their own native state. Also, the presence of protein
fibrils has been related to the appearance and development of more than 20 neurodegenerative
and non-neurodegenerative diseases, known as amyloid diseases. Hence, a profound knowledge
about the fibrillation mechanisms and the structure of the resulting protein assemblies is required
to develop new therapeutical strategies, but also to exploit the special structural properties of
such protein aggregates in new technological fields.
In this way, the kinetics of HSA fibril formation under different solution conditions and the
structures of resulting fibrillar aggregates were determined. It was observed that fibril formation is
largely affected by electrostatic shielding: At physiological pH, fibrillation is more efficient and
faster in the presence of up 50 mM NaCl. In contrast, under acidic conditions a continuous
progressive enhancement of HSA fibrillation is observed as the electrolyte concentration in
solution increases. The fibrillation process is accompanied by a progressive increase in β-sheet and
unordered conformations at the expense of α-helix one as revealed by CD, FT-IR and tryptophan
fluorescence spectra. The presence of structural intermediates in the aggregation pathway, such
as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates suggest that
fibril formation takes place by the competent-association of these aggregates. The resultant fibrils
are elongated but curly, and differ in length depending on solution conditions. In addition, very
long incubation times lead to a complex morphological variability of amyloid fibrils, such as long
straight fibrils or flat-ribbon structures, amongst other. In addition, the formations of ordered
aggregates of amyloid fibrils, such as spherulites, which possess a radial arrangement of the fibrils
around a disorganized protein core, or fibrillar gels were also observed. All these observations lead
to the conclusion that HSA can be used as a suitable model to study protein fibrillogenesis.
On the other hand, the utility of the obtained HSA fibrils as building blocks to construct new and
useful nanomaterials as tested. We reported a method to obtain metallic Au nanowires by using
lysozyme protein fibrils as bioscaffolds to generate a complete gold coating layer on the
biotemplate surface by the attachment of gold seeds and subsequent overgrowth of the coating
layer by sequential addition of gold salt growth solution (seeded-mediated mechanism). We
decided to use lysozyme fibrils since these display a straight and uniform core, which is more
suitable for the generation of nanowires. To obtain full coverage of the protein fibril, suitable
12

[Au]/[protein] molar ratio and number of sequential additions of Au ions must be chosen. The
hybrid metallic fibrils have been proved to be useful as catalytic substrates, provided their great
catalytically activity when incorporated in the reduction reaction of p-nitrophenol to p-
aminophenol by NaBH4. On the other hand, we also reported a method to obtain magnetic Fe3O4
nanowires by using HSA and lysozyme protein fibrils as bioscaffolds to generate a magnetic Fe3O4
coating layer on the biotemplate surface by the in situ controlled Fe nanoprecipitation method.
The magnetic properties of the magnetic nanowires have been tested by using a superconducting
quantum interference device (SQUID), and their potential applicability as MRI contrast agents was
analysed by means of magnetic resonance imaging at different Fe doping concentration in
phantoms.
13

Contents
1.1
1.2
1.3
1.4
CHAPTER 1
SYNTHETIC AND NATURAL POLYMERS
1.2.1
1.2.2
1.2.3
1.3.1
1.3.2
1.3.3
1.1.- Introduction
Introduction
Classification of polymers
Synthetic polymers
Biopolymers
Proteins
(Bio)polymer structure
Primary structure
Secondary structure
Tertiary structure
Molecular self-assembly
Since many years ago, polymeric molecules have progressively gained a key role in human´s
life and activity. Many examples about the use of polymers can be easily found in our daily
activity in different fields such as clothing, health care, packing or waste water treatments;
from the early times, humans have taken advantage of polymeric materials in the form of
wood, leather, natural gums and resins, cotton fibres, wool and silk (1).
Polymeric molecules found in Nature also possess very important biological functions as in the
case of nucleic acids, proteins or carbohydrates. These biomacromolecules take part in
different biochemical processes and are necessary in anabolic and catabolic processes;
furthermore, they fulfill an essential structural role during the development and growth of
living organisms as evidenced, for example, in plants, which are mainly built from cellulose
fibres, or animals, composed of linear protein materials such collagen in skin, sinew and bone,
myosin in muscles, and keratin in nails and hair. Polymers are giant molecules built up by
repetitive covalent unions of small chemical units known as monomers. Polymers usually have
high molecular weights and, for this reason, are also called “macromolecules”. They can be
classified in two main groups regarding their origin: synthetic and biological ones. In general,
15
16
17
18
19
19
20
20
22
23
15

synthetic polymers are obtained from processes developed in laboratories while biological
polymers (biopolymers) are found in nature, but many of them can be also synthetically
synthesized (1)-(3).
From a historical perspective, first attempts to obtain lab-synthesized polymers dated from
1868 with the synthesis of cellulose nitrate by the chemical modification of natural polymers
(pyroxin made from cotton and nitric acid with camphor). However, in 1909, Leo Hendrick
Baekeland synthesized a plastic material, the bakelite, which can be considered as the true
first synthetic polymer. From 1920s, different polymers of commercial importance were
synthesised; however, the main interest was focused on controlling their chemical
composition along the synthetic process, and no cautions about a suitable control of the final
molecular structure were considered since the synthesis of macromolecules was an empirical
work. In the middle of 20 th century, chemists and physicists provided new methods to establish
the structural description of polymeric molecules. In this regard, Herman Staudinger´s, a
German chemist who introduced the concept of macromolecule, and W. T. Carother´s, an
American chemist who classified the polymers according to their preparation method (i.e.,
addition and condensation polymers), works deserve special attention.
From a general point of view, polymer molecules show considerable flexibility due to rotation
around molecular bonds. In solution, the shape of these macromolecules alters continuously
due to thermal energy so they can be treated as random coils, even when the rotation around
molecular bonds does allow complete flexibility, while steric and excluded volume effects
oppose the formation of this kind of configuration. For these reasons, dissolved linear polymer
molecules will tend to be more extended than random coils. In addition, polymer-polymer and
polymer-solvent interactions exert an important effect in polymer chain configurations and
must be taken into account. If segments of the polymer chains stick together, then a tighter
configuration than random coil will result, enabling aggregate formation or precipitation (3).
1.2.- Classification of polymers.
Polymers can be classified in many different ways. The most obvious classification is based on
the origin of the polymer; i.e., natural polymers (biological macromolecules) such as proteins,
celluloses or natural gums, and synthetic polymers (non biological polymers). Overall, non-
biological polymers are formed by flexible chains with a relatively “low” number of monomers
16

in contrast to biological polymers, which consist of a larger number of monomers and with
stiffer chains.
1.2.1.- Synthetic polymers.
Synthetic polymers can be classified taking into account their physical and chemical
characteristics, molecular structure, polymerisation mechanisms, chemical composition,
building blocks or thermal behaviour. Among all of them, synthetic polymers have been
classified into two classes according to their preparation method: i.e., condensation and
addition polymers. For condensation polymers, the reaction occurs between two
polyfunctional molecules by eliminating a small molecule, for example, water. In contrast,
addition polymers are formed in a chain reaction of monomers which have double bonds in
their molecular structure.
According to polymer structure, it is possible to divide synthetic polymers into three wide
categories: linear, branched (non-linear) and cross-linked polymers. When the structural units
are repeated in only one dimension, linear polymers are obtained. Their molecular structure is
shown in Figure 1.1a. On the other hand, when the monomeric units are repeated in two or
three dimensions through covalent bonds, branched, cross-linked or network polymers are
obtained. In the case of branched polymers (also known as graft copolymers), single side-
branched chains are connected to polymer backbones during the synthesis, and which remain
singly dispersed (see Figure 1b). For cross-linked polymers, adjacent linear chains are
chemically or physically connected to two or more polymer backbones serving as “bridges” to
reticulate the polymeric system (see Figure 1.1c).
Figure 1.1. a) Linear, b) branched, and c) cross-linked polymers.
17

On the other hand, in terms of chemical composition, polymers can be classified as
homopolymers and copolymers. Homopolymers are those polymers composed of only one
type of structural unit, whereas copolymers posses two or more different monomers.
Copolymers can be additionally classified considering the sequential and structural order of
their constituent building blocks (see Figure 1.2):
Random copolymer: the building segments in the backbone follow a random sequence.
Alternate copolymer: the segments that configure the copolymer show a specific
alternating sequence.
Block copolymer: the polymer backbone is formed by the covalent bonding of large
sequences of different homopolymers (each one forming a monomer).
Graft copolymers: the copolymer molecule posses several segments grafted along the
polymeric backbone.
Figure 1.2. Schematic representation of different copolymers: (a) Random copolymer (b)
alternating copolymer, (c) and (d) block copolymer, (e) and (f) graft copolymer.
1.2.2.- Biopolymers.
Natural polymers are vital for the development, growth and perpetuation of any living
organism. Under this consideration, the most important ones are nucleic acids, proteins and
polysaccharides. The building units of these biological polymers are nucleotides, amino acids,
and carbohydrates, respectively. Among all these types, the present work is focused on the
study of proteins since they are an essential constituent of all organisms and virtually
participate in every process within cells.
18

1.2.3.- Proteins.
The most versatile macromolecules in living organisms are proteins. These play an important
and crucial function in almost all biological processes, and are formed by the linkage of
repetitive structural units, called amino acids, through peptides bonds (4). These are covalent
chemical bonds formed between two amino acids, in which the carboxyl group of one
molecule reacts with the amino group of the other, as shown in Figure 1.3. Thus, polypeptide
chains have two different terminal groups: on one side, the polypeptide chain posses an amino
group with positive polarity and, on the other, a carboxylic group with negative polarity. By
convention, the amino group has been considered the beginning of the polypeptide chain.
Figure 1.3. Peptide bond formation between the amino group of one amino acid and the
carboxyl group of the other.
1.3.- (Bio)polymer structure.
It is known from elemental organic chemistry that the physical state of a homologous series of
any type of chemical compound changes as their molecular size increases. From the low to the
high molecular weight end of the molecular spectrum, the members of a series change
progressively from the gaseous state, passing through liquids of increasing viscosity
(decreasing volatility) to low melting solids and, ultimately, terminate in high-strength solids.
Polymers belong to the high molecular weight end of the molecular spectrum, which provides
them very interesting and useful properties such as unusual crystallinity, a wide range of
thermal and mechanical behavior, and high resistance to chemical degradation. All these
properties are related to the chemical and structural aspects of polymers, which can be
divided into three different levels:
1. The monomer chemical structure (primary structure)
2. The single polymer chain (secondary level)
3. Aggregation of polymer chains (tertiary structure)
19

Additionally, to obtain a better knowledge about the macromolecular structure, it is necessary
to study the molecular forces existing between polymer chains, such as ionic and covalent
bonds, dipole-dipole interactions, hydrogen bonding, induction forces or van der Waals
interactions, amongst others (5).
1.3.1.- Primary Structure.
The primary structure refers to the atomic composition and chemical structure of the
monomers, which compose the polymeric chains. The electrical and chemical properties of
polymeric chains are much related to the nature and chemistry of the constituent monomers
and the kind of bonds established between them. Also, the macromolecular size of the
polymer is responsible of their physical and mechanical properties.
1.3.2.- Secondary Structure.
To understand the properties of polymers, it is necessary to develop a physical picture of what
these long molecules are really like. The secondary structure is a real physical description of
the polymer, and describes the size and shape of a single individual macromolecule. The
polymer molecular architecture can be influenced by both the nature of monomers and how
they are bonded each other. In order to have a suitable description about the molecular
architecture of a polymer, it is necessary to define two important terms: configuration and
conformation.
Configuration refers to the relative position of the atoms in a polymeric chain, which is
determined by the chemical bonds. The mechanical and thermal properties of polymers are a
function of chain configuration; thus, the configuration of a polymer cannot be altered unless
these bonds are broken. The different possible configurations of polymers which have the
same stoichiometric molecular formula but different structural formulation are called isomers.
There are two main geometrical configurations of polymers: cis and trans. These structures
cannot be changed by physical means (e.g. rotation). The cis configuration originates when
substituent groups are located at the same side of a carbon-carbon double bond. Trans
configuration refers to the case when the chemical substituents are on opposite sides of the
double bond (see Figure 1.4a,b). On the other hand, taking into account the position of the
atoms in a polymer chain three main configurations are observed: atactic polymers, in which
the substituent groups have a random orientation (below and/or above) along the polymeric
backbone; isotactic polymers, where the substituent groups are in the same plane along the
20

main polymeric chain; and sindiotactic polymers, in which the substituent groups are in an
alternating opposite orientation along the polymer backbone (see Figure 1.4c-e).
Figure 1.4. Schematic representation of the polymers configuration a) cis, b) trans, c) polymer
atactic, d) isotactic polymer, and e) sindiotactic polymer.
Molecular conformation refers to the special spatial orientation of a macromolecule. To
modify the macromolecular conformation it is not necessary to break out covalent bonds, but
it is only necessary to rotate the atoms around single bonds in the polymer backbone.
Therefore, the conformation of a polymeric molecule can be changed, for example, by
application of thermal energy or by interactions between the macromolecules and the solvent
molecules. The great number of possible conformations of macromolecules depends on
different factors such as polymer crystallinity, steric effects or their physical state (i.e., if the
polymer is in solution or in its molten or solid state), with the constraint that macromolecules
tend to adopt a minimum energy conformations state. Typical macromolecular conformations,
as random coil, fully extended chain, folded chain or helix are depicted in Figure 1.5. The
random coil is an asymmetric conformation characterized by a random orientation of
macromolecular segments. It is typical of polymers in solution, of molten polymers and solid
amorphous polymers. All other conformations reported in Figure 1.5 are symmetric and,
21

therefore, characteristic of solid crystalline polymers. Hence, when the concept of
macromolecular conformation is limited to a single macromolecule, it defines the polymer`s
secondary structure.
Figure 1.5. Secondary structure of polymers.
1.3.3.- Tertiary Structure.
Macromolecular conformation related to several macromolecules arranged together in any
liquid or solid phase defines the polymer´s tertiary structure. Usual tertiary structures are
depicted in Figure 1.6. These can be represented either by a tight arrangement of random coil
chains (spaghetti-like structure) as in molten or amorphous solid polymer; by a fringed-micelle
structure as observed in semi-crystalline solid polymers; or in regularly folded chains or super-
helix tertiary structures as in the case of highly crystalline solid polymers.
Figure 1.6. Tertiary structure of polymer.
Any polymeric material in the solid state posses great amounts of polymeric molecules in the
form of aggregates. The process of molecular aggregation can lead to the formation of either
crystalline or amorphous materials, which depend on both the polymer molecular structure
22

and the available configurations of the polymeric backbone´s building blocks. The ability of
polymer chains to establish a sufficient high number of dipole-dipole interactions, hydrogen
bonding, or van der Waals interactions, amongst others, is also a key factor in promoting and
controlling the molecular aggregation process in spite of the energy involved in this kind of
intermolecular forces is very weak (0.5-10 kcal/mol) if compared with covalent bonds (50-100
kcal/mol)
1.4.- Molecular self-assembly.
Molecular self-assembly is a spontaneous process by which molecules interact with each other
to form well-ordered supramolecular structures. The self-association between molecular
building blocks is mediated through non-covalent association of molecules (molecular
recognition processes). In this way, extraordinarily complex assemblies are formed in various
natural systems, and synthetic preformed molecular building blocks are assembled together by
keeping a delicate balance between non-covalent repulsive and attractive interaction by using
forces such as van der Waals and ionic interactions, metal coordination, H-bonding, π-π
interactions or hydrophobic forces. This process has motivated many different studies in the
fields of nanotechnology and materials science based on the design of new functional
nanostructures (6)(7). The only requirement for self-assembly is that the building block should
contain moieties able to establish non-covalent interactions in specific environments (6)(7).
Thus, advanced self-assembled nanomaterials have already been prepared from many
different types of molecules such as lipids, synthetic peptides, nucleic acids, low molecular
weight surfactants, dendrons, transition metals, inorganic nanoparticles or polymers (8)(9).
The latter have recently received great attention due to their increasingly scientific importance
and vast utility in a wide range of technological applications by their ability to spontaneously
self-organize into well-ordered aggregates, either in the solid state or in semidilute and dilute
solutions (9)(10).
Nowadays, the self-organization of molecular building blocks is probably one of the most
prominent topics of modern sciences. Indeed, supramolecular self-assembly is the key strategy
of Nature for fabricating multifunctional structures such as enzymes, ribosomes, membranes
channel or transport proteins from rather simple molecules such peptides, sugars and lipids.
For instance, Nature produces complex intermolecular structures with biomolecules, through
the control of biomolecular primary, secondary, tertiary and quaternary structure. The
23

presence of electrostatic charge in biomolecular primary structure provides natural systems
with a molecular-level tool to control intramolecular conformation (secondary and tertiary
structure) and intermolecular aggregation (quaternary structure), such as the association of
lipids to form cellular membranes, the protein folding mechanisms to construct functional
peptides and proteins, or the supramolecular organization of relevant biological entities such
as enzymes, ribosomes or transport channels.
In order to mimic the self-assembly processes observed in Nature (10)(11), block copolymers
have become of great scientific importance because of their unique and associative properties
as a consequence of their molecular structure. In particular, synthetic block copolymers can
self-organize spontaneously into unprecedented morphologies (which are related to their
segmental incompatibility), either in solid state or in semidilute or dilute solutions. For
instance, in bulk block copolymers made of thermodynamically incompatible segments phase-
separate into organized discrete nanophases, which can be exploited in many applications
ranging from impact-resistant materials to nanolithography and nanoelectronics. Similarly, in
dilute solutions block copolymers lead to the formation of colloidal aggregates such as
polymeric micelles or polymer vesicles, which have an enormous potential for applications in
cosmetic, the food industry, medicine and biotechnology.
In solution, the main attraction forces leading to block copolymer self-assembly are:
hydrophobic and electrostatic interactions, and hydrogen bonding. In this context, these
macromolecules can be considered as building blocks. In a selective solvent, blocks that can be
solubilized in this solvent tend to get in contact with the main phase, whereas blocks that are
insoluble in the solvent tend to be hidden from the main phase, leading to the formation of
organizated aggregates such spherical micelles, cylindrical micelles or vesicles (12). However,
amphiphilicity is not the only driving force that can be exploited for creating aggregates in
solution. The pairing of complementary charges in the polymer backbone is also a vector for
self-organization. Hydrogen bonding interactions are also important for the development of
self-assembling supramolecular aggregates, in which monomeric units are reversibly bound via
weak non-covalent interactions to form well-ordered structures.
For example, due to the great current interest in the development and improvement of nano-
devices, many of these synthetic block copolymers are being used in nanopatterning using the
top-down and bottom-up fabrication methods (Figure 1.7). The top-up strategy uses physical
engineering tools for carving of materials with a considerable macro-size to obtain a small
24

nanodevice. During the process, the lateral dimension of the material is reduced until having a
nanostructured material as occurred, for example, in the case of silicon integrated circuits
fabricated by selective layer deposition. In contrast, the bottom-up strategy takes advantage
of the self-association properties of molecular blocks, which possess nanometric size. The
living cell is a complex construction given by the Nature, and it is the best example for the
bottom-up strategy (8)(13)(14).
Figure 1.7. Schematic representation of two both strategies used to get nanostructured
materials: a) Top-down and b) Bottom-up.
Top-down
Physical engineering
Bottom-up
Supramolecular self-assembling
Nanostructural
Material
On the other hand, peptides and proteins show also amphipathic properties. The
intramolecular fold of these macromolecules allows to show specific faces or display specific
regions toward the bulk solvent phase; in this way, it is logical to assume that these regions
have hydrophilic or hydrophobic nature. For instance, the cylindrical structure of the α-helix
contain a sequence of hydrophobic amino acids which are positioned on one side of the
cylindrical face, and another sequence of hydrophobic amino acids on the opposite face of the
cylindrical structure. For β-sheet structure, hydrophobic and hydrophilic amino acids of a
polypeptide chain can be ordered in an alternating way. In this manner, the side lateral chains
of the amino acids are on opposite faces into the β-sheet structure (8).
25

Contents
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.2.1
2.2.2
2.2.3
2.3.1
2.3.2
2.4.1
2.4.2
2.9.1
2.11.1
2.11.2
2.11.3
2.11.4
2.11.5
Introduction
Surface tension
Wilhelmy plate method
Monolayers and Langmuir-Blodgett films
Axisymetric drop shape (ADSA)
Light scattering
Static light scattering (SLS)
Dynamic light scattering (DLS)
Electron microscopy
Transmission electron microscopy (TEM)
Scanning electron microscopy (SEM)
Atomic force microscopy (AFM)
Fluorescence spectroscopy
Circular dichroism (CD)
Infrared spectroscopy
Rheology
Viscoelasticity
X-ray diffraction
CHAPTER 2
Methods
Additional experimental techniques used in fibrillogenesis
studies
UV-Vis spectroscopy
Polarized light optical microscopy
Emission spectroscopy
Superconducting quantum interference device (SQUID)
Magnetic resonance imaging (MRI)
Laboratory equipment
28
28
28
29
31
32
36
40
44
44
46
47
48
52
54
58
59
64
72
72
75
77
78
80
82
27

2.1.- Introduction
There are many analytical techniques suitable for the characterization of synthetic and
biological macromolecules. This chapter is a brief summary of the basic theoretical background
and experimental setups used to perform the different experiments discussed in this Thesis.
In aqueous solutions of amphiphilic self-assembled systems, the aggregation process mostly
depends on concentration or/and temperature. The most usual experimental tools to follow
their self-assembly process into structural aggregates (micelles, vesicles...) are surface tension,
fluorescence spectroscopy, light scattering or nuclear magnetic resonance, amongst others. On
the other hand, the structural characteristics of the resulting self-assembled nanostructures
can be determined by techniques such as X-ray diffraction, light scattering or electron and
atomic microscopies.
2.2.- Surface tension
The critical micelle concentration (cmc) of amphiphilic systems and, in particular, in block
copolymer solutions can be determined by measuring their surface tension () as a function of
concentration. Surface tension depends on polymer concentration below its cmc and,
therefore, such behavior is denoted by a fairly sharp decrease in a vs. log (c) plot. There are
several methods for determining the surface tension of amphiphilic polymer solutions such as
the Du Noüy ring, the Wilhelmy plate, the pendant drop, the bubble pressure or the sessile
drop methods. The most common ones are the Du Noüy ring and Wilhelmy plate techniques
(15).
2.2.1.- Wilhelmy plate method
The Whilhelmy plate method measures the force (F) with which a platinum plate of known
perimeter (L= l+d) is pulled downward by an interface, in our particular case the air/water
and air/chloroform interfaces. The surface tension force, Lcos , is equal to the weight of the
liquid meniscus adsorbed onto the plate and detected by a balance (16), which can be
expressed through the following equation:
2.01
28

where w is the meniscus’ weight detected by the balance, is the contact angle defined by the
meniscus shape on the wet platinum surface plate, and l is the width and d the plate thickness,
respectively (Figure 2.1). Considering very small and the plate thickness negligible compared
with its width, the former expression is simplified to:
Figure 2.1. Illustration of a fully wet Wilhelmy plate in contact with an aqueous solution.
2.2.2.- Monolayers and Langmuir-Blodgett films
Many insoluble substances such as long chain fatty acids, alcohols, surfactants and polymers
can be spread from a solvent onto water to form a one-molecule thick film, called monolayer.
Molecules in a monolayer can be arranged in different ways, especially when they are closely
packed, depending on the lateral forces between them (17). The obtention of surface
pressure-area isotherms is one of the main methods used to study monolayer formation.
Surface pressure, , is the reduction of the surface tension due to the presence of the
monolayer: , where 0 is the surface tension of the solvent (usually water), and
the surface tension of the system. Surface pressure-area isotherms are often measured for
films/monolayers compressed using a Langmuir trough (Figure 2.2).
In general, an insoluble substance is spread from a volatile solvent to ensure the formation of a
uniform monolayer, and its area is controlled through a moveable barrier. The horizontal force
necessary to maintain the float at a fixed position is measured using a torsion balance, which
provides the surface tension values. Since only micrograms of materials are present in
2.02
29

monolayers, it is necessary to ensure that the water is as pure as possible (17). On the other
hand, it is also possible to deposit the monolayer film onto a solid substrate, or build up a
multilayer by successive depositions of monolayer films. In this case, this technique is called
Langmuir-Blodgett and the films are named Langmuir-Blodgett (or LB) films. A monolayer is
adsorbed homogeneously with each immersion or emersion step, thus films with very accurate
thickness can be formed. This thickness is accurate because the thickness of each monolayer is
known and can therefore be added to find the total thickness of a Langmuir-Blodgett film.
These LB films are of great interest since they enable the construction of functional multilayer
stacks which can be used in molecular electronic devices, such as gas sensors, diodes or in
semiconductor devices as insulators in transistors.
Figure 2.2. Scheme of a Langmuir trough.
When a monolayer is compressed on the water surface, several phase transformations can be
observed. These changes are almost analogous to those present in bulk phase matter, where
the most common states are gas, liquid and solid. The phase changes may be readily identified
by monitoring the surface pressure as a function of the area occupied by the film (surface
pressure-area isotherm) (18). This is the two dimensional equivalent of the pressure-volume
isotherm for bulk materials (gas/liquid/solid). Figure 2.3 shows the surface pressure-area
isotherm plot of a hypothetical long chain organic monolayer material (e.g., a long-chain fatty
acid). This diagram displays most of the characteristic features observed for long chain
compounds (18). The shape of the surface pressure-area isotherm depends on the lateral
interactions between molecules. These, in turn, depends on the molecular packing, which is
influenced by factors such as the size of the hydrophilic group, the presence of polar groups,
the length and number of hydrocarbon chains, and their conformation.
Briefly, in the gaseous state (G) the molecules are far enough apart on the water surface so
30

that they exert little force one on another. As the surface area of the monolayer is reduced,
the hydrophobic chains of the molecules will begin to interact. The liquid state formed is
generally called the expanded monolayer phase (E). At this stage, molecules at the water
surface are in a random rather than in a regular orientation, with their polar groups in contact
with the subphase. As the molecular area is progressively reduced, a condensed (C) phase may
appear. In the condensed monolayer state the molecules are closely packed and are oriented
with the hydrophobic groups pointing away from the water surface.
Figure 2.3. Surface pressure-area isotherm for a chain organic monolayer.
2.2.3.- Axisymetric drop shape analysis (ADSA)
Axisymetric drop shape analysis (ADSA) is a drop shape technique for measuring the interfacial
surface tension, γ. This method can be performed on the measured profiles of captive bubbles,
sessile drops, pendant drops or bubbles. The principle of the technique is based on the
determination of the drop shape by the balance between: i) the force of gravity which deforms
the drop and ii) the surface tension which resists the deformation by minimizing the drop
surface area (17). The relationship between these two forces is described by the Laplace
equation (19):
The Laplace equation is written in terms of the surface tension, , the principal radii of the
drop, and , and the pressure difference across the curved interface, . The pressure
drop has two contributions, , where is the pressure difference at a
2.03
31

eference plane, and the term gz is an additional pressure which arises from the gravitational
component at any point on the surface. This is given by the surface density difference, ,
between the bubble and the continuous phase, and its vertical distance from the reference
plane (17).
ADSA technique allows to determine both the surface area, , as well as the surface tension, .
Additionally, both quantities enable the derivation of important surface rheological properties,
such the elasticity or the surface dilatational modulus, E, of a film, defined as
. This can also be defined as a complex function written as
, where E’ and E’’ correspond to the storage and loss moduli, respectively, by analogy
with 3D rheology. Assuming the mechanical properties of the adsorbed layer follow Maxwell’s
behavior, the storage and loss moduli can be written as (20)-(22)
2.04a
2.04b
where E0i and 0i ( are the corresponding weights of the i-th relaxation element
to the total elasticity modulus, E0, and the characteristic relaxation frequency, respectively,
and 0i is the characteristic relaxation time of the Maxwell model.
2.3.- Light scattering
Scattering is a general physical process in which a form of radiation, such as, sound or a
moving particle, are forced to deviate from its straight trajectory by one or more localized non-
uniformities in the medium through it propagates. In the case of light, it is fairly simple to
understand the origin of its scattering considering it as an electromagnetic wave. Light will
interact with the charges inside a given molecule remodelling their spatial charge distribution.
The quantification of this effect is reported by a certain physical quantity, the polarizability (α).
The charge distribution follows the time-modulation of the electric wave vector of the incident
light beam and, therefore, the molecule constitutes an oscillating dipole or electric oscillator.
32

This oscillating dipole acts as an emitter of an electromagnetic wave with the same wavelength
as the incident one (the scattering process is elastic), which is emitted isotropically in all
perpendicular directions to the oscillator, as illustrated in Figure 2.4. The angle of observation
with respect to the direction of the incident light beam is called the scattering angle, and it
provides a measure of the accessible length scales by a light scattering experiment (23).
Figure 2.4. Oscillating dipole induced by an incident light wave, and emitting light (23).
For molecules or particles equal or larger than /20, being the wavelength of the incident
radiation, several of these oscillating dipoles are created simultaneously within one given
particle. As a consequence, some of the emitted light waves possess a significant phase
difference. Accordingly, interference of the scattered light emitted from such individual
particles leads to a non-isotropic angular dependence of the scattered light intensity. The
interference pattern of intra-particular scattered light, also called the particle form factor, is
characteristic of the geometry of the scattering particle. Hence, this provides a quantitative
means for the structural characterization of particles in very dilute solutions by light scattering.
On the other hand, for particles smaller than /20, only a negligible phase difference exists
between light emitted from various scattering centers within a given particle; in this case, the
detected scattered intensity will be independent of the scattering angle, and it only will
depend on the particle mass, which is proportional to the total number of scattering centers
one particle contains. The difference in the interference pattern of light scattered by small and
large particles, is illustrated in Figure 2.5 (23).
So far, we have considered light scattering as a purely elastic process where the emitted light
has exactly the same wavelength as the incident light. Particles in solution, however, usually
show a random motion (Brownian motion) caused by thermal density fluctuations of the
solvent. As a consequence of temporal changes in inter-particle positions and the
corresponding temporal concentration fluctuations, both the interference pattern and the
resulting scattered intensity detected at a given scattering angle also change with time,
33

eflecting the Brownian motion of the scattering particles (23). This phenomenon provides the
basis of dynamic light scattering, an experimental procedure which yields a quantitative
measure for the mobility of scattering particles in solution, characterized by their self-diffusion
coefficients. Most modern particle size analyzers used to determine the size of particles in
solution are based on this principle, further commented in detail below (23)-(25).
Figure 2.5. Interference pattern of light scattered from small particles (left) and large particles
(right). For simplification, only two scattering centers are shown (23).
General standard light scattering setups consist of the following components (Figure 2.6) (23)-
(26):
1. The incident light source, typically a laser source. This provides coherent and
monochromatic light with an intensity between some few milliwatts (mW) and several watts
(W). In practice, the light intensity needed for a successful scattering experiment depends on
the sensitivity of the optical detector and on the scattering power of the sample itself, as
determined by its size, concentration, and refractive index increment. As shown in Figure 2.6,
typically, the primary laser beam is guided and focused onto the sample by optical mirrors and
lenses. The laser beam diameter within the sample is well below 1 mm, which defines the
scattering volume.
2. The light scattering cell, in most cases a cylindrical quartz glass cuvette of outer
diameter between 10 and 30 mm, embedded within an index matching and thermostating
bath. Using an index matching bath around the cylindrical light scattering cuvette is important
to suppress unwanted diffraction effects of the incident and the scattered light at the sample-
air interfaces.
3. The detector, either a photo-multiplier tube or a very sensitive avalanche photo diode
34

(APD) and its associated optics (pinhole or optical fiber) mounted on the arm of a goniometer.
The detector optics determines the horizontal and vertical dimensions of the scattering
volume, whereas its depth is defined by the incident laser beam width. Band pass filters with
high transmission at the incident laser light wavelength are often used in front of the detector
to suppress undesired contributions of stray light or fluorescence from the sample.
4. The electronic hardware components associated with the detector used for signal
processing. The scattered intensity I (q,t), which is monitored by the optical detector, is
typically digitalized and stored in a computer. These raw data have to be further processed: in
the case of static light scattering, particle form factor models can be used to fit the
experimental data; in a dynamic light scattering experiment, the raw data have to be
converted into the time intensity correlation function g2 (q, τ). This is done during the
measurement using a very fast hardware correlator as, for example, the ALV 5000 from ALV,
Langen, Germany.
5. In a typical light scattering experiment, light from a laser source passes through a
polarizer, which defines the polarization of the incident beam and, then, enters the scattering
medium. The scattered light, then, passes through an analyser, which selects a given
polarization and finally enters a detector. The position of the detector defines the scattering
angle, θ. In addition, the intersection of the incident beam and the beam intercepted by the
detector defines a scattering region of volume V. In modern light scattering experiments the
scattered light spectral distribution is measured by a photomultiplier, which is the main
detector (24).
Figure 2.6. Schematic representation of an usual light scattering experiment (26).
35

2.3.1.- Static light scattering (SLS)
As mentioned above, matter scatters electromagnetic waves due to the induction of an
oscillating electromagnetic dipole which serves as a source for the scattered light wave. In this
way, Rayleigh scattering is described in terms of three factors: the incident light, the particle
(i.e. a macromolecule), which serves as an oscillating dipole, and the scattered light. The
schematic model is shown in Figure 2.7. The equation for the electric field of incident polarized
light beam at x position and at time, t, may be expressed by the following well-known equation
(27)-(32):
where E is the electric field or electric intensity, E0 is the amplitude of the incident electrical
wave, ν is the light frequency in solution, and t is the propagation time.
Figure 2.7. Sketch of a light scattering process (23).
If an incident light reach a particle (i.e. macromolecule), whose size is smaller than the
wavelength , d

where r is the distance of the dipole from the observer (from the scattered light sample to the
detector) and is the angle between the dipole axis and the line r. The division by the square
of the light velocity, c 2 , is a dimensional correction. Substituting
equation:
2.07
in the above
Comparing the intensity I of the scattered radiation to the intensity I0 of the incident radiation,
which is proportional to the square of its amplitude E0:
This equation is called the Rayleigth equation for plane polarized light. The scattering intensity
increases rapidly as the light wavelength decreases. The intensity also depends on the angle :
there is no radiation along the direction ( =0) in which the dipole vibrates.
Let us consider a system of volume V containing N independent identical scattering particles.
Then, the total intensity I of scattered light is:
Generally, the Rayleight scattering equation is directly applicable to gases, where molecules
move randonly. In liquids, there exists fluctuations in particle concentration inside a volume
element, which results in fluctuations of the polarizability α. Therefore, to apply the Rayleigh
scattering equation to the liquid state, it is necessary to account for these fluctuations by using
α. In this way, since the polarizability α depens on the dielectric permitittivity ε, and
correspondingly on the index of refraction n, then:
2.08
2.09
2.10
2.11
37

For a dilute solution, polarizability α may be written as:
where dn/dc is the differential refractive index and is an experimentally measurable quantity.
Substitution of the equation 2.12 into equation 2.10, and considering that
expressed as
number:
Defining the Rayleigh ratio and is often designed as R:
which is independents of r and . Simplifying:
Then the light scattering becomes:
where
2.12
can be
, where M is the molecular weight and NA is the Avogadro’s
.
As mentioned above, for very dilute solution of small particles (for example, nanoparticles or
polymer chains of size smaller than /20) the scattering intensity is independent of the
scattering angle, and the density fluctuation from the surrounding solvent can be substracted
by considering the excess Rayleigh ratio, R ( ; then, the scattering
intensity only depends on the scattering power of the dissolved particles (i.e., from their
molecular mass) and the osmotic pressure. For non ideal solutions, and after several
theoretical considerations (3), the scattering equation can be rewritten:
2.13
2.14
2.15
2.16
38

When , the former equation can be simplified to the so-called Rayleigh-Gans-Debye
relation:
Actually, ΔR cannot be experimentally measured since I0 and r are unknown during the
experiment. However, in a routine experiment ΔR is determined from the experimentally
measured scattered intensities of the solution, Isolution, and a reference substance, Iref (usually
benzene or toluene) (23) which enables the derivation of their respective Rayleigh ratios.
Hence, the following equation can be derived providing a relation between the molecular
weight, Mw, of the particle considered and the second virial coefficient, A2 (29)-(30):
where
,
and
2.17
2.18
2.19
. I, Is and Iref, are
the experimentally measured scattered intensities of the solution, solvent, and reference,
respectively. For small particles at dilute concentrations equation 2.19 can be reduced to:
On the other hand, for large scattering particles the scattered intensity is no longer
independent of the scattering angle. The scattering vector q, which is experimentally
determined by the scattering angle and the laser light wavelength, , provides a quantitative
measure of the length scale of the static light scattering experiment, given by:
For very dilute solutions, interference between different scattering particles, the so-called
2.20
2.21
39

structure factor, can be neglected. In this case, the angular dependence of the measured
scattered intensity I(q) is only caused by intraparticle interference. For dilute and semidilute
solutions, to take into account the effects of particle concentration and solute-solvent
interactions on the measured scattering intensity, the Zimm equation can be used:
In order to determine Mw, Rg, and A2 it is necessary to prepare several dilute solutions of
different concentration and measuring the scattering intensities data at various scattering
angles (27)(28).
2.3.2.- Dynamic light scattering (DLS)
Light scattered by a set of mesoscopic particles produces a random interference pattern on a
screen. This pattern, generally possess the form of irregularly spaced and sized bright spots
called speckles. The scattered light intensity pattern remains unchanged as long as the
particles do not change their position. Particle motion naturally leads to a temporal evolution
of the scattered speckle field since one interference pattern is continuously replaced by
another. In a single speckle spot, this evolution is observed as strong temporal intensity
fluctuations, with a certain well-defined temporal correlation. The intensity fluctuations are
inherently related to the scatterers’ dynamics and, therefore, the temporal correlation
function depends on the particles shift. Thus, the measurable correlation properties of light
can be linked to the dynamical properties of particles which, in turn, can provide information
about their flow velocity and direction, particle size, density of moving scatterers...
In dynamic light scattering, also known as photon correlation spectroscopy (PCS) or quasi-
elastic light scattering (QELS), the diffusive motions of particles in solution gives rise to
fluctuations in the scattered light intensity on the microsecond timescale. This technique is
one of the most popular methods used to determine the particles size by measuring the
temporal fluctuations of the scattered light intensity. Roughly, this is made by focusing a
monochromatic light beam, such a laser, on a solution with particles in Brownian motion; this
causes a Doppler shift when the light “hits” the moving particle, changing the light wavelength.
This change is related to the size particle (27)(32).
In the scope of DLS, temporal fluctuations are usually analyzed by means of the intensity
2.22
40

autocorrelation function (ACF). In the time domain, the correlation function usually decays
with time (Figure 2.8), and faster dynamics leads to faster decorrelation of the scattered
intensity trace. It can be shown that for a random process the intensity ACF is the Fourier
transform of the power spectrum and, therefore, DLS measurements can be equally well-
performed in the spectral domain. In fact, DLS experiments were initially discussed in terms of
the broadening of the spectrum peak of monochromatic light due to Doppler shifts
experienced by propagating light waves scattered by moving particles (31).
Figure 2.8. Illustration of autocorrelation function (31).
To detect the intensity fluctuation with time, a DLS system requires an autocorrelator on top
of a regular SLS system, as shown in Figure 2.9.
Figure 2.9. Sketch of a dynamic light scattering system. The pulse amplifier discriminator
converts the analogic signal of the photodetector, I(t), in a digitalized signal, which is further
processed by the autocorrelator into the autocorrelation function.
41

As commented above, Figure 2.10a illustrates how the intensity (I) varies with time (t). I(t)
fluctuates around its mean value, . It may appear completely random and, therefore,
meaningless, but it is not. Motions of particles (i.e. polymer molecules) and solvent molecules
contribute to the change of I(t) with time. This apparently noisy signal carries the information
about particles motions.
Figure 2.10. a) Light scattering intensity I(t) fluctuates around its mean value . b)
Autocorrelation function is obtained as the long-time average of
for various delay times . The aurocorrelation function decays from to over time. The
amplitude of the decaying component is .
The autocorrelator calculates the product average of two scattering intensities I(t) and I(t+)
measured at two different times separated by , the delay time. The average product
is a function of and is called the correlation function of I(t), or the intensity-
intensity correlation function. The correlator converts I(t) into . Hence, the
intensity-intensity correlation function is the average of over a long period T.
Hence, assuming that the long time average is equal to the ensemble average, we can write:
Note that, if the system is at equilibrium, the ensemble average does not change with time
2.23
42

and, therefore, . The autocorrelation function of I(t) (Figure 2.10a)
is shown in Figure 2.10b. When = 0, . With increasing ,
decays to an asymptotic level (baseline), .
Because the scattering intensity I(t) fluctuates around a mean value , it is convenient to
separate its fluctuating component, , as . By definition, .
Then, the correlation function can be rewritten as:
The autocorrelation of is zero upon increases of . When ,
2.24
. The decaying componet in is . The
initial height of the decaying component is . Division of by
leads to the intensity autocorrelation function as:
where
normalized intensity autocorrelation function:
2.25
(the coherent factor), and the second factor is the baseline-subtracted,
The coherent factor depends on the coherence of the light reaching the photodetector. The
measured intensity correlation function is related to the field correlation function by the
Siegert relation:
where is:
where is the electric field conjugate function. For monodisperse spherical particles:
2.26
2.27
2.28
43

where is the characteristic delay rate, which is related to the translational diffusion
coefficient, D, of a solute by means of the expression:
The diffusion coefficient is frequently used to determine the hydrodynamic radius, RH, of the
constituent particles by using the Stokes-Einstein equation:
where KB is the Boltzmann constant, T the absolute temperature and the liquid viscosity.
The hydrodynamic radius obtained by DLS represents an ideal hard sphere that diffuses with
the same speed as the particle under examination. Actually, particles are solvated and the
radius calculated from the particle diffusion corresponds to the size of the dynamic solvated
particle.
2.4.- Electron microscopy
Microscopy is an important tool for the characterization of nanomaterials structure. For
example, this set of imaging techniques may be used to examine the detailed shape, size and
distribution of a wide range of nanoscopic objects. There are different available classes of
microscopic techniques, such as the traditional optical microscopy, or more powerful ones
such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and
scanning probe microscopy (SPM), amongst others.
2.4.1.- Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) involves the transmission of an electron beam
through a sample in a high-vacuum environment. The images and their associated contrasts
arise from regional differences in electron densities in such sample. TEM has a resolution of ca.
1 to 100 nm and can provide very detailed structural information of polymeric materials. The
TEM specimens need to be very thin in order to enable the transmission of electron beams
through the sample.
2.29
2.30
2.31
44

A transmission electron microscope (Figure 2.11a) is constituted by an electron source
(electron gun) which emits electrons travelling through the vacuum created into the
microscope column. A condenser lens focus the electron beam on the sample, and several
objective lenses are used to form the diffraction pattern at the back focal plane and the
sample image at the image plane; there are also some intermediate lenses to magnify the
image or the diffraction screen. To obtain an improved contrasted image, an objective
diaphragm is inserted in the back focal plane to select the transmitted beam. At the bottom of
the microscope, the non-scattered electrons reach a fluorescent screen, which provides a
contrasted image of the specimen, with a darkness distribution corresponding to its different
electron densities.
Figure 2.11. Schematic diagram of electron microscopes: a) TEM; b) SEM .
In order to solve the material´s structure on the mesoscopic scale at even high resolutions
comparable to interatomic distances, high-resolution transmission electron microscopes
(HRTEM) emerge as a powerful tool. HRTEM uses the transmitted and scattered electron
beams directed to a sample to create a diffraction image, in contrast to conventional TEM that
only uses the transmitted beams. The high resolution image recorded can be interpretable
directly in terms of projections of individual atomic positions. In this way, defects and other
inhomogeneities present in a material specimen can be accurately determined. A great
advantage of the HRTEM microscope is the capability to observe both electron microscope
images and diffraction patterns for determined selected area electron diffractions from a
45

sample (33)-(35). The selected area electron diffraction in an electron microscope is formed by
setting an aperture in the imaging plane of the objective lens. Only rays passing through this
aperture contribute to the diffraction pattern at the far field. For a perfect lens without
aberrations, the diffracted rays come from an area that is defined by the back-projected image
of the selected aperture. The aperture image is typically a factor of 20 times smaller because
of the objective lens magnification. The smallest area that can be selected in SAED is limited
by the objective aberration (36).
2.4.2.- Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) is another valuable electron microscopy technique for the
examination and analysis of the microstructural characteristics of solid objects, with a
resolution of ca. 5 nm. SEM can also be used to obtain compositional information of
nanomaterials by coupling an electron diffraction X-ray scattering detector. SEM enables the
observation of heterogeneous organic and inorganic materials on the mesoscopic scale (37). In
a typical SEM experiment, an electron beam is focused to obtain a very fine spot size that is
rastered over the surface, and an appropriate detector collects the electrons emitted from
each point. In this way, an image having a great field depth and a remarkable three-
dimensional appearance is built up line by line. The specimen is usually coated with a
conducting film prior to examination to enhance electron conductivity and, thus, improving
image contrast.
A scanning electron microscope (Figure 2.11b) consists of an electron gun at the top of the
column, which creates a divergent electron beam. In the column, which is under vacuum, a
series of magnetic apertures focuses the electron beam, and an electrostatic field drives the
electrons through a small spot called crossover and accelerates them through the column until
the sample chamber, where the electron beam interacts with the sample. The signals resulting
from the beam-sample interaction are monitored. Finally, SEM constructs a virtual image from
the signal emitted from the sample by scanning the electron beam line by line through a
rectangular (raster) pattern on the sample surface. The scan pattern defines the area
represented in the image. At any time, the beam illuminates only a single point in the pattern.
As the beam moves, the signals it generates vary in strength, reflecting
structural/morphological differences in the sample.
46

2.5.- Atomic force microscopy (AFM)
An atomic force microscope (AFM) is part of a large family of instruments termed as scanning
probe microscopes (SPM). The common factor in all SPM techniques is the use of a very sharp
tip probe, which is scanned across a surface of interest. The interactions between the probe
and the surface are able to produce a high resolution image of the sample (potentially up to
the sub-nanometre scale) depending on the technique and sharpness of the probe tip. For
AFM, the probe usually interacts directly with the surface probing the repulsive and attractive
forces which exist between the probe and the sample surface to produce a high resolution
three-dimensional topographic image of the latter. The great versatility of AFM makes possible
measurements in air or fluid environments rather than in high vacuum, which allows the
imaging of polymeric and biological samples in their native states. In addition, it is highly
adaptable, with tip probes being able to be chemically functionalised to allow quantitative
measurement of interactions between many different types of materials (38).
Figure 2.12. Typical AFM setup. A tip probe is mounted at the apex of a flexible cantilever,
made of Si or Si3N4. The cantilever itself or the sample surface is mounted on a piezo-crystal,
which allows the position of the probe to be shifted respect to the surface. The deflection of
the cantilever is monitored by changes in the path of a laser light beam deflected from the
upper side end of the cantilever recorded by a photodetector (38).
An AFM instrument consists of a sharp tip probe mounted near the end of a cantilever arm,
which is able to make a full scanning across the entire sample surface. By means of monitoring
the arm’s deflection originated by the topographic features present on the sample surface, a
three dimensional picture can be built up at high resolution. In Figure 2.12 the basic setup of a
47

typical AFM is shown. Cantilevers commonly are either V-shaped or a rectangular “diving
board” shaped. The cantilever posses a sharp tip at its free end, which acts as the interaction
probe. This probe has most commonly the form of a square-based pyramid or cylindrical cone.
The probe is brought into and out of contact with the sample surface by the use of a piezo-
crystal where the cantilever or the surface itself is mounted, depending on the particular
equipment used. The movement in this direction is conventionally referred to as the Z-axis. A
laser light beam is reflected from the reverse side of the cantilever onto a position-sensitive
photodetector. The configuration of the photodetector consisted of a quadrant photodiode
divide into four parts with a horizontal and vertical dividing line. If each section of the detector
is labelled from A to D, as shown in Figure 2.12, then the deflection signal is calculated by the
signal difference detected in each part of the detector (38).
2.6.- Fluorescence spectroscopy
Once a molecule is excited by the absorption of a photon, this can return its ground state
through either fluorescence emission, internal energy conversion (i.e. direct return to the
ground state without fluorescence emission for example, by heat radiation), intersystem
crossing (possibly followed by phosphorescence emission), intramolecular charge transfer
and/or conformational change. All these processes can be visualized in the Perrin-Jablonski
diagram (Figure 2.13). The singlet electronic states are denoted as S0 (fundamental electronic
state), S1, S2, ... and the triplet states as, T1, T2, ..., with different vibrational levels associated
with each electronic state. It is important to note that energy absorption is very fast (≈10 -15 s)
with respect to all other processes (so that there is no concomitant displacement of the nuclei
according to the Franck-Codon principle) (39)(40). The vertical arrows corresponding to the
energy absorption process start from the 0 vibrational energy level, S0, since most of molecules
are in this state at room temperature. Absorption of a photon, hence, can bring a molecule to
one of the vibrational levels of S1, S2, ...
Emission of photons accompanying the S1 S0 relaxation is called fluorescence. It should be
emphasized that, apart from a few exception, fluorescence emission occurs from S1 and,
therefore, its characteristics (except polarization) do not depend on the excitation wavelength
(of course only one species exists in the ground state). The transition between the ground
state and the excited stated (0-transition) is usually the same for absorption and fluorescence.
However, the fluorescence spectrum is located at higher wavelengths than the absorption one
48

ecause of the energy loss in the excited state due to vibrational relaxation (Figure 2.14).
According to the Stokes rule the fluorescence emission wavelength should be always higher
than that of absorption. However, in most cases the absorption spectrum partly overlaps the
fluorescence spectrum, i.e. a fraction of light is emitted at shorter wavelengths than the
absorbed light. Such an observation seems to be, at first, in contradiction with the energy
conservation principle. However, such energy defect is compensated by the fact that a small
fraction of molecules is in a higher vibrational level in the ground state as well as in the excited
state at room temperature (39).
Figure 2.13. Perrin-Jablonski diagram and illustration of the relative positions of absorption,
fluorescence and phosphorescence processes (39).
In general, differences between the vibrational levels are similar in the ground and excited
states, so that the fluorescence spectrum often resembles the first absorption band. The gap,
expressed in wavenumber, between the maximum of the first absorption band and the
maximum of fluorescence is called the Stokes shift.
It should be noted that photon emission is as fast as photon absorption. However, excited
molecules remain in the S1 state for a certain time (a few tens of picoseconds to a few
hundreds of nanoseconds, depending on the type of molecule and its surrounding medium)
before emitting a photon or undergoing other relaxation processes. Thus, after excitation of a
49

population of molecules by a very short light pulse, the fluorescence intensity decreases
exponentially with a characteristic time, reflecting the average lifetime of the molecules in the
S1 excited state.
Figure 2.14. Illustration of the vibrational bands in the absorption and fluorescence spectra.
As a consequence of the strong influence of the local environment or surrounding medium on
fluorescence emission, fluorescent molecules are currently used for physicochemical,
biochemical and biological investigation. For example, fluorescence spectroscopy can be
employed as a highly sensitive method for protein analysis. Intrinsic protein fluorescence
deriving from the naturally fluorescent amino acid tryptophan (and to a lesser extent from
tyrosine and phenylalanine) can provide information on the protein conformational structure
and it changes. On the other hand, the use of extrinsic fluorescent dyes offer additional
possibilities for protein characterization, i.e. to monitor their refolding and unfolding
processes, to detect molten globule intermediates, or to characterize protein aggregation and
fibrillation amongst others. For example, the dye 1-anilinonaphthalene-8-sulfonate (ANS) is
hardly fluorescence in aqueous environments but become highly fluorescent in apolar-organic
solvents or upon adsorption to solid phases. Other important dyes for protein characterization
are, for example, Nile Red (used for monitoring protein conformational changes), 9-
(dicyanovinyl)-julolidine (DCVJ, used to probe tubulin structure and the formation of
hydrophobic microdomains along protein aggregation), or Congo Red and Thioflavin T (used to
characterize the extent of -sheet formation along protein fibrillation) (40)(41).
50

Figure 2.15. Schematic representation of a spectrofluorometer (40).
Figure 2.15 shows the components of a conventional spectrofluorometer. The light source is
commonly a high-pressure xenon arc lamp, which offers the advantage of a continuous
emission from 250 nm to the infrared. A monochromator is used to select the excitation
wavelength. Fluorescence is collected at right angles with respect to the incident beam and
detected through a monochromator by a photomultiplier. Automatic scanning of wavelengths
is achieved by motorized monochromators, which are controlled by the electronic devices and
the computer, in which the data are stored (39)(40). The optical module contains various parts:
a sample holder, shutters, polarizers if necessary, and a beam splitter consisting of a quartz
plate reflecting a few per cent of the exciting light towards a quantum counter or a
photodiode. A quantum counter usually consists of a triangular cuvette, containing a
concentrated solution of a dye whose fluorescence quantum yield is independent of the
excitation wavelength.
51

2.7.- Circular dichroism (CD)
Circular dichroism (CD) spectroscopy is commonly used for the study of macromolecules
(synthetic and natural). CD technique is based on the rotation and absorption of left and right
circularly polarized light passing through a sample so < CD signal will be generated when the
circularly polarized light pass through a chiral sample. In this regard, a plane-polarised light
beam can be considered to consist of two equal and opposing left and right circularly polarized
light beams. In Figure 2.16, the electric vector of these beams is viewed looking down the
direction of the light source. When the magnitudes of these vectors are equal, their resultant
will trace the linear path of the electric vector amplitude of a plane-polarised light beam. This
means that the magnitudes of the vector will be no longer the same. This difference involves
that the resultant vector will be that of an elliptically polarized light beam passing through the
sample (40)(42):
Figure 2.16. Illustration of how circularly polarized light interacts with achiral and chiral
molecules. Plane polarized light can be considered as composed of the sum of two equal and
opposing left and right circularly beams. These views represent the electric vector component
of the beams looking down the direction of propagation of the light source. a) The resultant
transmitted beam is still present when the plane polarized light pass through an achiral
sample; b) circularly polarized light is present when it passes through a chiral sample; and c)
pictorial representation defining ellipticity (42).
In many biological systems, the presence of different asymmetric chemical groups around a
carbon atom originates chirality, as in the case of amino acids forming the polypeptide
52

ackbone of proteins. Since proteins, then, are built by units that are chiral, they will interact
with left and right circularly polarized light to different extents giving rise to circular dichroism
(40)(42). Hence, circular dichroism can be defined as the difference between the absorption of
left and right circularly polarized light by a chiral molecule:
where AL and AR are the absorption of the left and right circularly polarized light, respectively,
by the chiral molecule. For achiral molecules , and no CD signal is present. For chiral
molecules, at some wavelength, and the resulting will be non-zero; it can have
either a positive or negative value depending on the relative intensities of the left and right-
handed light absorbance. Usually, in the analysis of protein structures CD instruments scale the
chirality as ellipticity, , expressed in millidegrees. Therefore, a CD signal can be viewed either
as an absorption difference or as the produced ellipticity, although both are related each
other. However, ellipticity is not a useful quantity to compare different samples as its
magnitude differs depending on the amount of protein present in the sample. For CD spectral
data, the most useful units are delta epsilon the molar circular dichroism, Δ, and the mean
residue ellipticity []MRE (MRE).
Figure 2.17. Schematic representation of a typical circular dichroism spectrometer.
To obtain a circular dichroism spectrum (Figure 2.17), a periodic variation in the polarization of
the light beam is induced by the polarization modulator through all ellipticities. This polarized
light passes through the sample to a photomultiplier detector. If the sample is not optically
2.32
53

active, the light beam does not vary through this cycle. With the introduction of an optically
active sample, a preferential absorption is seen during one of the polarization periods, and the
intensity of the transmitted light now varies during the modulation cycle. The variation is
directly related to the circular dichroism of the sample at that specific wavelength. Successive
detection is performed at various wavelengths leading to the generation of the full CD
spectrum.
2.8.- Infrared spectroscopy
Molecules consist of atoms which have a certain mass and are connected by elastic bonds. As a
result, they can perform periodic motions, i.e. they have vibrational degrees of freedom. All
motions of the atoms in a molecule relative one each other are a superposition of the so-called
normal frequencies. These are described by a normal coordinate. For polyatomic molecules,
the number of characteristic frequencies is for non-linear molecules and for
linear molecules, where N is the number of atoms in the molecule, which define their
vibrational spectra. These spectra depend on atoms mass, their geometrical arrangement, and
the strength of their chemical bonds. Molecular aggregates such as crystals or complexes
behave like “super-molecules” in which the vibrations of the individual components are
coupled. As a first approximation the normal vibrations are considered not coupled, i.e. they
do not interact. However, the bonds elasticity does not strictly follow Hooke’s law. Therefore,
overtone and combinations of normal vibrations may appear (40)(43).
Infrared (and RAMAN) spectrometers are the most important tools for observing vibrational
spectra. Depending on the nature of the vibration, which is determined by the symmetry of
the molecule, vibration may be active or forbidden in the infrared (or RAMAN) spectrum.
From basic theory, it is easy to understand that a molecule can take up an amount of energy,
, to reach a vibrationally excited state only when the frequency of the incident radiation on
a molecule is the same as the vibrational frequency of the latter. This phenomenon involves
either a change of the molecule’s dipole moment or a change in its polarizability. If the former
phenomenon takes place, we will observe an IR band; if the second one occurs, we will
observe a RAMAN band. The absorption wavelength range of IR spectroscopy is from 2.5 to
1000 μm, which corresponds to the energy with = 4000-10 cm -1 , with the light
velocity.
54

According to classical mechanics, the vibrating atoms groups in a molecule can be modelled as
balls joined by springs (the molecular bonds). To take the simplest example, let us consider
two balls joined by one spring representing a diatomic molecule. In this approximation, two
atoms and the connecting bond are treated as a simple harmonic oscillator composed of 2
masses (atoms or balls). These two balls can vibrate with a characteristic frequency that
depends on the mass of the balls and on the spring force (spring force can be approximated by
Hooke’s law). Matematically, the equation
, describes the characteristic
oscillation frequency and how it depends on mass and spring constant force . According
to this equation, stiffer spring constant (stronger bonds) result in a higher frequency, whereas
heavier atoms result in lower frequencies. This simple system possess only a single
characteristic frequency. The balls can only vibrate with this characteristic frequency or an
integer multiple of this frequency. A more complex molecule can still be modelled quite
accuarately as balls of different massess joined by springs of different tensions (representing
different atoms with different bonds). Figure 2.18 shows a diagram representing the vibration
of a diatomic molecule or one vibrational degree of freedom of a polyatomic molecule, which
is excited by a transition from energy level to (40)(43)-(45).
Figure 2.18. Observation of the excitation of a vibrational state in the electronic ground state
by infrared absorption.
In the classical harmonic oscillator,
, where x is the displacement of the spring.
Thus, the energy or frequency is dependent on how far one stretches or compresses the
spring. If this simple model were true, a molecule could absorb energy of any wavelength.
However, vibrational motion is quantized and, therefore, the molecules can only exist in finite
energy states. In the case of harmonic potentials, these states are equidistant, i.e.
where the energy levels are numbered as ni. At ni = 0, the
potential energy has its lowest value, which is not the potential minimum energy (43)(44).
55

According to the selection rules, only transitions to the next energy level are allowed;
therefore, molecules will absorb an amount of energy equal to . This rule is not inflexible
and, occasionally, transitions of , , or higher are observed. These correspond to bands
called overtones in an IR spectrum. They are of lower intensity than the fundamental vibration
bands.
As mentioned above, infrared spectra cover the near infrared(0.75-3 m), middle infrared (3-
30 m) and far infrared (30-300 m) range. In this region, most molecules show absorption or
emission bands arising from symmetry-allowed interaction with the radiation. The energy of
the absorbed and emitted light is equivalent to the energy difference between the lowest
vibrational states of the electronic ground state of a molecule.
Three types of instruments for IR absortion measurements are commonly available: dispersive
spectrophotometers with a grid monochromator, Fourier thansform spectrometers employing
an interferometer, and non-dispersive photometers using a filter or absorbing gas (typically
used for analysis of atmospheric gases at specific wavelengths).
Until 1980s, the most widely used instruments for IR measurements were dispersive
spectrometers. Nowadays, this type of instruments has been displaced by Fourier transform
(FT) spectrometers because of their speed, reability, enhanced signal-to-noise ratio. Infrared
spectrometers (Figure 2.19) usually combine a radiation source, collimator, fixed mirror,
moving mirror, a sample holder, a device for spectral dispersion or selection of radiation
(beam splitter), and a radiation detector, all connected to appropiate recording and evaluation
facilities. An ideal spectrometer records completely resolved spectra with a maximum signal-
to-noise ratio. It requires a minimum amount of sample which is measured nondestructively in
a minimum time, and it provides facilities for storing and evaluating the spectra. FTIR
spectrometers can be single-beam or double-beam instruments. A typical procedure for
determining transmittance or absorbance with this type of instrument is to first obtain a
reference interferogram by scanning a reference (usually air) twenty or thirty times coadding
the data, and storing the results in the computer. A sample is inserted in the radiation path
and the process repeated. The ratio of the sample to reference spectral data is, then,
computed to obtain the transmittance at various frequencies.
Infrared spectroscopy (IR) has long been used to determine molecular structures and to
identify unknown compounds. IR data have been used to obtain information about the
56

chemical composition, configuration, and crystallinity of polymeric and biological materials
(43). In particular, FTIR spectroscopy is a versatile spectroscopic technique for analyzing
protein structure in solution, in aggregates and inclusion bodies. Most IR studies focus on the
characterization of protein secondary structures by using absorption bands reflecting the
motions of peptide groups. Infrared spectra of proteins may be obtained either in the solid
state or in solution. The vibrational spectra of proteins exhibit several relatively intense peaks
which shift in wavenumber and intensity from one sample to another. There are several
prominent bands for the study of polypeptides and proteins; however in this study, we will
focused partularly on the amide bands, which are of particular interest for the study of protein
conformation: the amide I, between 1680 and 1600 cm -1 , is mainly associated with the
stretching vibration of the C=O bond; amide II, found between 1580 and 1480 cm -1 , and amide
III band, observed between 1300 and 1200 cm -1 , are both associated with coupled C-N
stretching and N-H bending vibrations of the peptide groups, respectively. These bands are
conformationally sensitive, and their position may be attributed to a dominat protein
conformation, that is, α-helix, parallel and antiparallel β-sheets, turns or random coil (38)(42).
In particular, the following vibrational frequencies had been assigned to: 1654 cm -1 for α-helix;
(1624, 1631, 1637, and 1675 cm -1 for β-sheets; 1663, 1670, 1683, 1688, and 1694 cm -1 for
turns; and 1645 cm -1 for other structures (43)(46)-(48). The relative amounts of the four
secondary structures are determined by the relative areas under the band peaks.
Figure 2.19. Scheme of a FTIR instrument.
57

2.9.- Rheology
Rheology involves the study of the deformation and flow of matter due to compressive
stresses acting on a material. Particularly, it refers to the behaviour of materials when a
mechanical force is applied on. Rheology includes three main concepts such as force,
deformation and time. Irreversible flows, reversible elastic deformations or their combination
(viscoelasticity) can, therefore, model and describe a rheological phenomenon under certain
assumptions. The type of deformation depends on the state of matter; for example, gases and
liquids will flow when a force is applied, whilst solids will deform by a fixed amount and, then,
back to their original shape when the force is removed. In the case of polymers, the rheological
and mechanical properties affect the polymers molecular properties, such as their molecular
weight, molecular weight distribution, conformation, architecture and crystallinity (49)(50).
Figure2.20 Schematic diagram of basic tool geometries for a rotational rheometer: a)
concentric cylinder, b) cone and plate, c) parallel plate (50).
A typical rheometer measures the velocity of displacement of the moving surface and the
force exerted on one of the surfaces. Most rheometers are based on rotary motion and use
one of the three following geometries (Figure 2.20): concentric cylinder, cone and plate, and
parallel disk. In most cases the same rotary instrument can use all three of these flow
geometries. To generate the needed motion, they typically use actuators like a hydraulic
piston or ball screws found in standard tensile testing machines for solids. Solenoids or other
electromechanical actuator are often used for small amplitudes and low forces. There are two
basic designs of rheometers: controlled stress ones, where the stress is applied electrically via
a motor measuring the strain; and controlled strain instruments, in which a strain is imposed
and the stress is computed from the deformation of a calibrated spring system (50).
58

2.9.1.- Viscoelasticity
Many materials can be classified as solid or fluids, displaying elastic and viscous behaviour
respectively. Viscoelastic materials such as polymers combine the characteristics of both
elastic and viscous materials depending on the experimental time scale. Application of a stress
of relatively long duration may cause some flow and irrecoverable deformation, while a rapid
shearing would induce an elastic response in some polymeric fluids. Then, a classification of
these materials should include a consideration of the timescale of the measurement relative to
the characteristic time of the material. This ratio is given by the Deborah number (De): when De
is 1 the material will display both viscous and elastic behaviour and is described as viscoelastic,
whilst, for De » 1 and De « 1 the material has solid-like and viscous-like behaviour, respectively
(49)(50).
Pure elastic solid behaviour may be exemplified by a Hookean spring, and pure viscous flow
can be exemplified by the behaviour of a dashpot, which is essentially a piston moving in a
cylinder of Newtonian fluid. Nevertheless, the viscoelasticity cannot be described accurately by
neither spring nor dashpot alone, but a combination of both. In this way, several mechanical
models have been developed to describe the viscoelastic response of a given material. All of
these mechanical models are, then, supported on the combination of these two simple
elements (spring and dashpot), which represented the elastic behaviour of a solid body, and
the viscous flow for a liquid, respectively. Among all models, Maxwell’s and Kelvin-Voigt’s
models are the most frequently used (Figure 2.21) (50).
a) b)
E
Figure 2.21. a) Maxwell’s and b) Voigt’s models.
The use of mechanical models such as the spring and dashpot as analogues of the behaviour of
real materials enables us to describe very complex experimental behaviours using a simple
combination of models. The uses of models to represent a wide range of deformation are not
restricted to the application of shear stress; however, in this section, we will focus on the shear
properties of the material (1).
E
59

To overcome the poor description of real polymeric materials by either the spring or the
dashpot, Maxwell suggested a simple series combination of both elements (Figure 2.21a).
Because the material possesses the ability to flow, some inertial relaxation will occur and less
force will be required upon time to sustain the deformation. The goal in the Maxwell’s model
is to calculate how the stress varies with time, or expressing the stress in terms of the constant
strain to describe the time-dependent modulus. Let us consider in the Maxwell’s model, in
which one spring is attached to one dashpot, as shown in Figure 2.21a. When a force is acting
on the spring downward at (in one dimensional flow), the stress-strain relation for the
spring (Hookean material) may be described by (50):
where is the applied stress, is the strain, and G the elastic modulus. Conversely, the stress
response of the dashpot (a purely viscous Newtonian fluid) to an applied deformation rate may
be described as:
where
2.33
2.34
is the strain rate and , defines the viscous response of the dashpot with
Newtonian behaviour. In other words, the spring purely exhibits an elastic effect (as a Hookean
solid) and the dashpot exhibits purely a viscous effect (as a viscous fluid).
In the Maxwell’s element, both the spring and the dashpot support the same stress and,
therefore:
where and are the stresses on the spring and dashpot, respectively. However, the
overall strain and the strain rates are the sum of the elemental strain and strain rates,
respectively, that is:
or
2.35
2.36
60

where is the total strain rate, while
and
2.37
are the strain rates of the spring and
dashpot, respectively. Therefore, the total strain of the spring and dashpot at any time is the
sum of that of the spring and of the dashpot. Then, for a Maxwell’s model the strain rates can
be written as:
By rheometry we can be measure the response of a material to an oscillating stress or strain,
so it is considered as mechanical spectroscopy. When a sample is constrained in, for example,
a cone and plate assembly, an oscillating strain at a given frequency can be applied to the
sample. After an initial start-up period due to a transient sample state a stress develops in
direct response to the applied strain. If the strain has an oscillating value with time the stress
must also be oscillating. We can represent these two wave forms as in Figure 2.22. The elastic
and viscous effects are out of phase one each other by some angle (1).
Figure 2.22. An oscillating strain and the stress response for a viscoelastic material.
All the information about the response of the sample at this frequency is contained within
these wave forms. However, this information is not in a particularly tractable form. What we
would really prefer is to have a few representative terms such as the relaxation time and
2.38
61

elasticity or viscosity of the sample in order to characterize its material properties. In order to
obtain this information some mathematical manipulation is required. Two key features can be
utilized. The first feature is the maximum stress, , divided by the maximum strain, , which is
constant for a given frequency . This ratio is called the complex modulus :
is the radial frequency, which is , where is the applied frequency measured in Hz. The
other feature constant with time at any given frequency is , the phase difference (expressed
in radians). These two values, and , are characteristic of the material. It is straightforward
to visualise the situation where an elastic solid is placed in a cone and plate geometry. When a
tangential displacement is applied to the lower plate a strain in the sample is produced. That
displacement is transmitted directly through the sample. The upper cone will react
proportionally to the applied strain to give a stress response. An oscillating strain will give an
oscillating stress response that is in phase with the strain, so will be zero. However, if we
have a Newtonian liquid, the peak stress will be out of phase by
2.39
rad as the peak stress is
proportional to the strain rate. So, it follows that if we have a viscoelastic material some
energy is stored and some dissipated, and the stored contribution will be in phase whilst the
dissipated or loss contribution will be out of phase respect to the applied strain.
In order to describe the material properties as a function of frequency we need to use the
constitutive equation 2.30. This equation describes the relation between the stress and the
strain. However, it is most convenient to express the applied sinusoidal wave in the
exponential form of the complex number notation:
Now, the stress response lags by the phase angle :
So, substituting the complex stress and strain into the constitutive equation for a Maxwell fluid
the resulting relation is given by:
2.40
2.41
62

Using equations 2.40 and 2.41 in equation 2.42 and rearranging we have:
Thus, arrangement of this expression gives the complex modulus and frequency:
or:
where
2.42
2.43
2.44
2.45
is the characteristic relaxation time. This expression describes the variation of
the complex modulus with frequency for the Maxwell model. It is normal to separate the real
and imaginary components of this expression. This is achieved by multiplying by ( ) to
give:
where is an in phase elastic modulus with energy storage in the periodic deformation,
and is called the dynamic storage modulus. is an out of phase elastic modulus associate
with the energy dissipation as heat, and is called the dynamic loss modulus. Then:
These expressions describe the frequency dependence of the stress with respect to the strain.
2.46
2.47
2.48
63

It is normal to represent them as two moduli that determine the component of stress in phase
with the applied strain (storage modulus) and the component out of phase by 90 0 . In an
experiment, the amplitudes of the oscillation input ( ) and output ( ), and the phase angle
( ) are measured. Therefore, each oscillatory shear flow measured at a given provides two
independent quantities, amplitude ratio and phase angle:
The storage and loss moduli are subtle descriptions of the material properties of the system.
These two properties are related to the phase angle and complex modulus. These are both
functions of the applied frequency and represent an alternative description of the system.
The phase angle changes with the frequency, from 90 degrees at low frequency, to 0 degrees
at the high frequency limit; thus, as the frequency increases the sample becomes more elastic,
and the phase difference between the stress and the strain reduces.
2.10.- X-ray diffraction
X-ray diffraction (XRD) is a versatile, powerful and non destructive technique that reveals
detailed information about the chemical crystallographic structure of natural and
manufactured materials. Diffraction effects are observed when an electromagnetic radiation
impinges on periodic structures with geometrical variations on the length scale of the radiation
wavelength. The inter-atomic distances in crystal and molecules is around to 0.15 – 0.5 nm,
which correspond in the electromagnetic spectrum with the wavelength of X-rays, having
photon energies between 3 and 8 keV (3).
There are three different types of interactions between X-rays and matter. First, the
photoionization, where electrons may be liberated from their bound atomic states during the
process, and which falls into the group of inelastic processes. In addition, other inelastic
2.49
2.50
2.51
64

scattering process that incoming X-ray beams may undergo upon interaction with matter is the
energy transfer to an electron but, in this case, without a release of the electron from the
atom (phenomenon known as Compton scattering). Finally, X-rays may be elastically scattered
by electrons, process known as Thomson scattering. In this process, the electron oscillates at
the frequency of the incoming beam so the wavelength of the scattered radiation is conserved.
There are many theories and equations to correlate the diffraction pattern and the material
structure. The Bragg law is a simple way to describe the diffraction of X-rays by a crystal. In
Figure 2.23a, the incident X-rays reach the crystal planes with an incident angle θ and are
diffracted at an angle θ. The diffraction peak is observed when the Bragg condition is satisfied
(3)(51):
where λ is the wavelength of the incident X-rays, d is the distance between each adjacent
crystal plane (d-spacing), θ is the Bragg angle at which a diffraction peak is observed, and n (1,
2, ...) is an integer number, called the reflection order; this means that the Bragg condition can
be satisfied by various X–ray wavelengths. The Bragg law gives a simple relationship between
the diffraction pattern and the crystal structure, and many X-ray diffraction applications can be
easily explained by this law.
Figure 2.23. a) Incident and reflected X-rays form an angle θ which is symmetric to the crystal
normal plane; b) The diffraction peak is observed at a Bragg angle θ (52).
In X-ray diffraction using a single wavelength, the Bragg equation is typically expressed with n
= 1 for the first order of diffraction because higher order reflections can be considered as
2.52
65

esulting from different lattice planes. For instance, the second-order reflection from (hkl)
planes is equivalent to the first-order reflection from (2h, 2k, 2l) planes. The diffraction peak is
displayed as diffracted intensities at a range of 2θ angles. For perfect crystals, the peak is a
delta function, the dark straight vertical line shown in Figure 2.23b (52).
Figure 2.24. Diffraction patterns from crystalline solids, liquids, amorphous solids, and
monoatomic gases as well as their mixtures (52).
A typical diffraction peak is a broadened peak displayed by the curved line in Figure 2.23b. The
peak broadening can be due to many effects, including imperfect crystal conditions, such
strain, mosaic structures, and finite sizes; ambient conditions, such as atomic thermal
vibrations; and instrumental conditions, such as X-ray beam size, beam divergence, beam
spectrum distribution, and detector resolution. The curved line gives a peak profile, which is
the diffracted intensity distribution of the Bragg angle. The highest point on the curve gives the
66

maximum intensity of the peak, Imax. The width of the peak is typically measured by its full
width at half maximum (FWHM). The total diffracted energy can be measured by the area
under the peak, which is referred to as integrated intensity. The integrated intensity is a more
consistent value for measuring the diffracted intensity of a reflection since it is less affected by
all peak broadening factors (51)(52).
The Bragg diffraction condition is based on the existence of a long periodicity of crystalline
materials. As mentioned above, X-rays diffraction can provide information on the atomic
arrangement in material with long-range order, short-range order, or no order at all, like gases,
liquid, and amorphous solids, and a material may have one or to be a mixture of the former
atomic arrangement types. Figure 2.24 gives a schematic comparison of the diffraction
patterns for crystalline solids, liquids, amorphous solids, and monatomic gases as well as their
mixtures. The diffraction patterns shown in this Figure are displayed as diffracted intensity
versus assuming that the diffracted intensity is a unique function of the diffraction angle.
The diffraction pattern from crystals has many sharp peaks corresponding to various crystal
planes based on the Bragg law. The peaks at low angles are from crystal planes of large d-
spacing and, viceversa, at high angles. To satisfy the Bragg condition at all crystal planes,
the crystal diffraction pattern is actually generated from polycrystalline materials or powder
materials. Therefore, the diffraction pattern is also called powder diffraction pattern. A similar
diffraction pattern can be collected with a single crystal if this has been rotated at various
angles during data collection so that the Bragg law can be satisfied when the crystal is at the
right orientation.
Both amorphous solid and liquid materials do not have the long-range order of crystals, but
their atomic distances have a narrow distribution because of atoms are tightly packed. In this
case, the intensity of the scattered X-rays forms one or two maxima with a very broad
distribution in the range. The intensity vs. distribution, thus, reflects the distribution of
the atomic distances. In the case of a gas there is no order at all, i.e. the atoms are distributed
randomly in space. The scattering curve is featureless except that the scattered intensity drops
continuously with the increase of . On the other hand, the diffraction pattern from a
material containing both amorphous and crystalline features has a broad background from the
amorphous phase and sharp peaks from the crystalline phase. For example, many polymer
materials have an amorphous matrix with crystallized regions. The diffraction pattern may
contain air-scattering background in addition to sharp diffraction peaks. The air-scattering can
be generated from the incident beam or diffraction beam. If the air-scattering is not removed
67

y the diffractometer, the diffraction pattern contains a high background at low angle and
the background gradually decreases with increasing angle (52).
On the other hand, X-ray diffraction phenomena can also be explained in reciprocal space by
the reciprocal lattice and the Ewald sphere. Reciprocal lattice is a transformation of the crystal
lattice in real space to reciprocal space. The shape and size of a unit cell in real space can be
defined by three vectors, a, b, and c, all starting from any single lattice point. The unit cell of
the corresponding reciprocal lattice is, then, given by three vectors a*, b*, and c* (also
referred to as reciprocal lattice axes), and:
where V is the volume of the crystal unit cell in the real space and
Since each reciprocal lattice axis is the vector product of two lattice axes in real space, these
are perpendicular to the plane defined by the two lattice axes. The original lattice axes and
reciprocal lattice axes maintain the following relations:
and
Figure 2.26 illustrate the relationship between the original lattice in real space and the
reciprocal lattice. The unit cell of the original lattice is drawn in dotted lines. The three
reciprocal lattice axes define a unit cell of the reciprocal lattice (solid lines). The origin of the
reciprocal lattice axes, denoted by O, is the origin of the reciprocal lattice. The repeat
2.53
2.54
2.55
2.56
68

translation of the reciprocal lattice unit cell in three dimensions forms the complete reciprocal
lattice. Except the origin, each lattice point is denoted by a set of three integers (hkl), which
are the number of translations of the three reciprocal lattice axes, respectively, to reach the
lattice point. In other words, the vector drawn from the origin of the lattice point (hkl) is given
by:
and the direction of the vector is normal to the lattice planes (hkl) in real space. The
magnitude of the vector is given by the d-spacing of the (hkl) planes:
Figure 2.26. Relationship between the original lattice in real space and the reciprocal lattice
(49).
Therefore, each point (hkl) in the reciprocal lattice represents a set of lattice planes (hkl) in the
real space lattice. The position of the point in the reciprocal lattice defines the orientation and
d-spacing of the lattice planes in the real space lattice. The farther away a reciprocal lattice
point is from the origin, the smaller is the d-spacing of the corresponding lattice planes. For
example, the reciprocal lattice point (111) represent the (111) lattice planes in the real space
lattice, and the lattice vector is given by and
.
2.57
2.58
69

Figure 2.27. The Ewald sphere and Bragg condition in reciprocal space.
On the other hand, the relationship between the Bragg condition and the reciprocal lattice can
be explained visually by the Ewald sphere, also referred as reflection sphere. Ewald came up
with a geometrical construction to help the visualization of which Bragg planes are in the
correct orientation to diffract. In Figure 2.27, the diffracting crystal is located in the center of
the Ewald sphere, C. The radius of the Ewald sphere is defined as
. The incident beam can
be visualized as the vector from I to C, and the diffracted beam is the vector from C to P. Both
the incident and diffracted beams form an angle θ from a set of crystal planes (hkl). The d-
spacing of the crystal planes is dhkl In the Ewald sphere; both incident beam vector
diffracted beam vector
and
start at the poin C and end at the point O and P, respectively. The
vector from O to P is the reciprocal lattice vector and is perpendicular to the crystal
planes. The three vectors have the following relationship:
and their magnitude can be expressed based on the Bragg law as:
The point O is the origin of the reciprocal lattice, and the point P is the reciprocal point (hkl).
The Bragg condition is satisfied only when the reciprocal lattice point falls on the Ewald sphere.
2.59
2.60
70

For a single crystal, the chance to have a reciprocal lattice point on the Ewald sphere is very
small if the crystal orientation is fixed. Multiplying both ends of equation 2.59 by the three
lattice axes in real space, respectively, the Laue equations are obtained:
2 .61
The Laue equations establish that a periodic three-dimensional lattice produces diffraction
maxima at specific angles depending on the incident beam direction and the wavelength. The
Laue equations are suitable to describe the diffraction geometry of a single crystal. The Bragg
law is more conveniently used for powder diffraction. Both the Bragg law and Laue equations
define the diffraction condition in different formats.
On the other hand, the distance between the origin of the reciprocal lattice O and the lattice
point P is reciprocal to the d-spacing. The largest possible magnitude of the reciprocal lattice
vector is given by
. This means that the smallest d-spacing satisfying the Bragg condition is
. In powder X-ray diffraction, the random orientation of all crystallites can take all possible
orientations assuming an infinite number of crystallites. The trace of the reciprocal lattice
points from all crystallites can be considered as a series of spherical surfaces with origin O as
the center point. Therefore, the condition to satisfy the Bragg law is only if the d-spacing is
greater than half of the wavelength. In other words, the Bragg condition can be satisfied if a
reciprocal lattice point falls in a sphere of from the origin O. This sphere is called the
limiting sphere for powder diffraction.
X-ray diffraction systems have a variety of configurations and component options to fulfil
requirements of different samples and applications. As shown in Figure 2.25, a typical XRD
system normally consist of five basic components:
The X-ray source produces X-rays with the required radiation energy, focal spot size,
and intensity;
The X-ray optics establish the primary X-ray beam to the required wavelength, beam
focus size, beam profile, and divergence;
The Goniometer and sample stage establish and tune the geometric relation between
71

primary beam, sample, and detector;
The sample alignment and monitor assist users about sample positioning into the
instrument and monitors the sample state and position;
The area detector intercepts and records the scattering X-rays from a sample, and it
saves and displays the diffraction pattern into a frame.
Each of the former basic components may have several options suitable for various application
and functions. The whole system is controlled by a computer equipped with a software for
instrument control, data acquisition and data analysis. In addition to the five basic
components, there are some other accessories, such as a low and high-temperature stages,
helium or vacuum beam path for SAXS, beam stop, and alignment and calibration fixtures.
Figure 2.25. Scheme of a X-ray diffractometer.
2.11.- Additional experimental techniques used in fibrillogenesis studies
2.11.1.- UV-Vis absorption spectroscopy
Spectra are observed as the result of the energy exchange between a substance and the
electromagnetic radiation. If the energy is absorbed by a substance from the radiation field,
we have absorption spectra; if the energy is added to the radiation field, emission spectra are
observed. An electromagnetic radiation may be characterized by the frequency , the
wavelength , or the wave number . They are related as ,
velocity of light (39)(40)(53).
; where c is the
The three most important types of optical spectroscopies are ultraviolet and visible (UV-Vis),
fluorescence and infrared (IR) spectroscopy. They differ only in the selection of the incident
light wavelength. Both UV-Vis and fluorescence describe the phenomenon of electron
72

excitation; namely, a valance electron of a molecule is excited upon absorbing energy from the
electromagnetic radiation and is, thereby, transferred from one energy level to other more
energetic level. The spectra are electronic. In contrast, IR spectra describe the vibration of
atoms (not electrons) around a chemical bond, as previously described in certain detail.
An electron transition consists of the promotion of an electron from a molecular orbital in the
ground state to an unoccupied orbital by absorption of a photon. The molecule is, then, said to
be in an excited state. Let us recall first the various types of molecular orbitals. A orbital can
be formed either from two s atomic orbitals, from one s and one p, or from two p atomic
orbitals having a collinear symmetry axis. The bond formed in this way is called a bond. A
orbital is formed from two p atomic orbitals overlapping laterally. The resulting bond is called a
bond. For example, in ethylene (CH2=CH2) the two carbon atoms are linked by one and one
bond. Absorption of appropriate energy can promote, for example, one of the electrons to
an anti-bonding orbital denoted by . The transition is, then, called .
A molecule may also possess non-bonding electrons located on heteroatoms such oxygen or
nitrogen. The corresponding molecular orbitals are called orbitals. Promotion of a non-
bonding electron to an anti-bonding orbital is possible, and the associated transition is
denoted by . Hence, molecules containing a non-bonding electron, such as oxygen,
nitrogen, sulphur, or halogens, often exhibit absorption in the UV region. To illustrate these
energy levels, Figure 2.26 shows formaldehyde as an example with all their possible
transitions. In particular, the transition deserves further attention: upon excitation, an
electron is removed from the oxygen atom and goes into the orbital localized half on the
carbon atom and half on the oxygen atom. The excited state, thus, has a charge
transfer character, as shown by the increase observed in the dipole moment regarding the
ground state dipole moment of C=O (40)(53).
In summary, the electronic transitions observed in UV-Vis spectroscopy are ,
, , , , and . The energy of these electronic transitions is,
generally, in the following order: < < < < < . Of
the six transitions outlined, only the two lowest energetic ones ( and ) are
achieved by the energies available in the 200 to 800 nm. The last four types of electronic
transitions required higher energy inputs, below 200 nm, corresponding to the far ultraviolet
region of the electromagnetic spectrum. For instance, most transitions for individual
bonds take place below 200 nm and a compound containing only bonds is transparent (near
73

zero absorption) in the near-UV-Vis region. For example, methane (which has only C-H bonds)
the transition takes place at 125 nm.
In absorption and fluorescence spectroscopy, two important types of orbitals are considered:
the Highest Occupied Molecular Orbitals (HOMO) and the Lowest Unoccupied Molecular
Orbitals (LUMO). Both of these refer to the ground state of the molecule. For instance, in
formaldehyde, the HOMO is the orbital and the LUMO is the orbital.
Figure 2.26. Energy levels of molecular orbitals in formaldehyde and possible electronic
transitions (39).
When one of the two electrons of opposite spins (belonging to a molecular orbital of a
molecule in the ground state) is promoted to a molecular orbital of higher energy, its spin is, in
principle, unchanged so the total spin quantum number ( with
zero. Because of the multiplicities of both the ground and excited states ( ) are
equal to 1, both are called singlet states (usually denoted for the ground state) and , ,...
for the excited states (see Figure 2.27). The corresponding transition is called a singlet-singlet
transition. A molecule in a singlet excited state may undergo conversion into a state where the
promoted electron has changed its spin; therefore, there are two electrons with parallel spins,
and the total spin quantum number is 1 and the multiplicity is 3. Such state is called a triplet
state because it corresponds to three states of equal energy. According to Hund’s rule, the
triplet state has lower energy than the singlet state of the same configuration (39).
or
) is
74

Figure 2.27. Distinction between singlet and triplet states.
An instrument for measuring the absorption of UV and visible radiation is built of one or more
light sources, a wavelength selector, a sample container, radiation transducers, a signal
processors and readout devices. Figure 2.28 shows a scheme of a single-beam instrument for
absorption measurements. Single beam instruments vary widely in their complexity and
performance characteristics. The simplest ones consist of a tungsten bulb as the source, a set
glass filter for wavelength selection, a test tube for sample holders, a photovoltaic cell as the
transducer, and an analogmeter as the readout device. Other sophisticated instruments have
interchangeable tungsten and deuterium lamp sources, use a rectangular silica cell, and are
equipped with a high-resolution grating monochromator with variable slit. Photomultiplier
tubes are used as transducers, and the output is often digitalized, processed and stored in a
computer so that it can be printer or plotted in several forms (54).
Figure 2.28. General scheme of a single-beam instrument for UV-Vis absorption
measurements.
2.11.2.- Polarized light optical microscopy
Polarized light optical microscopy (POM) provides all the benefits of bright field microscopy
and yet offers a wide range of information, which is simply not available with any other optical
microscopy technique. POM can distinguish between isotropic and anisotropic materials. The
technique exploits the optical properties of anisotropy to reveal detailed information about
the structure and composition of materials.
75

Isotropic materials demonstrate the same optical properties in all directions of space. They
have only one refractive index and no restriction on the vibration direction of light passing
through them. In contrast, anisotropic materials have optical properties that vary with the
orientation of the incident light. They shown a range of refractive indexes depending both on
the light propagation direction through the sample and on the vibrational plane coordinates
(phenomenon known as birefringence). Birefringence is the term used to indicate the ability of
an anisotropic material to separate an incident ray into two rays, each polarized light ray
travels at two different speeds. One of the resulting polarized rays travels through the sample
with the same velocity in every direction and is termed the ordinary ray. The other ray travels
with a velocity that is dependent upon the propagation direction within the anisotropic
material. This light ray is termed the extraordinary ray. The two independent refractive indices
of anisotropic sample are quantified in terms of their birefringence defined as: ,
where and are the refractive indices of the extraordinary and ordinary rays, respectively.
This expression is true for any part or fragment of anisotropic material except when light
waves are propagated along the optical axis of the sample (55). The POM technique exploits
the interference of the splitted light rays as they are re-united along the same optical path to
extract information about these materials.
Birefringent specimens exhibit characteristic patterns and orientations of light and dark
contrast features that vary, depending on the shape and geometry of the object (linear or
elongate vs. spherical) and the molecular orientation. In the absence of a compensator,
spherical objects with radially symmetric molecular structure exhibit a dark upright
polarization cross superimposed on a disk composed of four bright quadrants (55).
POM is perhaps best known for its geological applications (primarily, for the study of minerals
in rock-thin sections), but it can be used to study many other materials. These include
minerals, composites, ceramics, fibers and polymers, and crystalline or highly ordered
biological molecules such as DNA, protein, starch, wood and urea.
As commented in detail previously, light can be represented as a transverse electromagnetic
wave made up of mutually perpendicular, fluctuating electric and magnetic fields. It means
that, at any point of a light beam, the magnetic field is always perpendicular to the electric
field, and oscillates in a plane perpendicular to the propagating direction of the light beam. In
plane polarized light, the electric field vector oscillates along a straight line of the propagation
light beam. Figure 2.29 shows a polarized light optical microscope configuration. In this
76

microscope, there are two polarized filters, the polarizer and the analyzer. The polarizer is
situated below the specimen stage, and the analyzer is situated above the objectives and can
be moved in and out of the light path as required. When both the analyzer and polarizer are in
the optical path, in a crossed configuration, no light passes through the system and a dark field
of view is present in the eyepieces.
Figure 2.29. Polarized light optical microscope configuration.
2.11.3.- Emission spectroscopy
Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) is a widespread technique
for elemental analysis of a sample (56). In Figure 2.30, a scheme of an ICP-AES instrument is
shown. A plasma source is used to make specific elements to emit light, after which a
spectrometer separates this light in their characteristic wavelengths. This technique is
especially suited for direct analysis of liquid samples. The sample solution is transformed into
an aerosol by a nebuliser. Small droplets (1-10 μm) are transferred by an argon plasma. When
the aerosol droplets enter the hot area of plasma, they are converted into salt particles. These
salt particles are split into individual molecules that will subsequently fall apart to atoms and
ions. In the plasma, even more energy is transferred to the atoms and ions, promoting the
excitation of their electrons. When these excited atoms and ions back to a lower excitation
state or to their ground state they will emit electromagnetic radiation in the ultra-violet/visible
range of the spectrum. The classical approach for ICP-AES measurement is to collect and
measure the emitted radiation radially, i.e. the optical axis is orthogonal to the central channel
of the ICP. The limit of detection is in de order of ca. ng/ml.
77

Figure 2.30. Scheme of an ICP-AES.
2.11.4.-Superconducting quantum interference device (SQUID)
SQUID is a sensitive device for measuring magnetic fields. SQUID magnetometers are used to
characterize materials when the highest detection sensitivity over a broad temperatures range
and application of magnetic fields up to several Tesla is needed. The main application of this
instrument is the study of magnetic properties of materials by measuring the induced or
remnant magnetic moment, usually as a function of applied magnetic field and temperature. A
SQUID magnetometer combines several superconducting components, including the proper
SQUID device, a superconducting magnet, detection coils, a flux transformer and
superconducting shields. To make a measurement, a sample is first attached to a sample rod.
The sample is then scanned through the center of a first-order or second-order
superconducting gradiometer. The gradiometers form a closed flux transformer that is coupled
to a SQUID. The shape and magnitude of the response curve can be then analyzed using a
computer to obtain a corresponding magnetic moment (57).
Figure 2.31. Scheme of a SQUID magnetometer (57).
78

In particular, we use SQUID to measure the magnetic properties of magnetic nanoparticles and
hybrids. In this regard, magnetic nanoparticles show a wide variety of unusual magnetic
properties as compared to the respective bulk materials. In a paramagnetic material, the
thermal energy overcomes the coupling forces between neighbouring atoms above the Curie
temperature, causing random fluctuations in the magnetization directions that result in a null
overall magnetic moment. However, in superparamagnetic materials the fluctuations affect
the direction of individual entire crystallites. The magnetic moments of individual crystallites
compensate each other and the overall magnetic moment becomes null. When an external
magnetic field is applied, the behaviour is similar to paramagnetic materials except that,
instead of each individual atom being independently influenced by an external magnetic field,
the magnetic moment of entire crystallites aligns with the magnetic field (Figure 2.32) (58)(59).
For magnetic nanoparticles, superparamagnetism occurs in magnetic materials composed of
very small crystallites (the threshold size depends on the nature of the material, i.e. Fe-based
materials becomes superparamagnetic at sizes

increasing temperature after cooling the sample in the applied magnetic field (Figure 2.33). At
temperatures well below the blocking temperature, the ZFC magnetization is small because
the sample is not in thermal equilibrium, and the magnetization directions of the particles in a
small applied field are mainly governed by the randomly oriented directions of magnetization.
With temperature increases, the smaller particles become superparamagnetic, and the
probability of finding a particle with magnetization directions close to that of the applied field
increases, resulting in an increase in the magnetization. With further temperature increases,
more and more particles become superparamagnetic, resulting in an increasing magnetization
until the net effect of the thermal energy becomes dominant and involves a decrease in the
magnetization value. In the FC state, the magnetization in the blocked state is preferably
frozen in a direction close to that of applied field. Therefore, the magnetization is considerably
larger than that in the ZFC state. The ZFC and the FC curves coincide above the bifurcation
temperature, which is the temperature above which all particles are superparamagnetic (59).
Figure 2.33. ZFC and FC magnetization curves measured on a ferrofluid material (59).
2.11.5.- Magnetic resonance imaging (MRI)
One of the most valuable and widely used imaging methods in medical diagnostic is the
Magnetic Resonance Image (MRI). Though the diagnostic potential of conventional MRI is
immense and a variety of different contrast models are well established, further
improvements of the method have been pursued in last years by application of magnetic
nanoparticles for contrast enhancement based on functional site specificity.
In MRI, the nuclear magnetic moment of protons is used as a sensitive probe of the chemical
neighbourhood of protons in different tissues and organs of human body. Nuclear moments
80

are aligned by means of an external magnetic bias field (commonly in the range of 0.2-3 T) and
the precession of the spins is excited by transverse RF pulses at a proton resonance frequency
of about 42.58 MHz T -1 . After applying the pulse sequence, the induced magnetization decays
and the longitudinal (T1) and transverse (T2) relaxation times of the precessing nuclear
magnetic moments show tissue-specific differences that are used to generate the required
image contrast. Imaging is performed by controlling external field gradients so that the
resonance condition is fulfilled only in a restricted local region and, then, by scanning the
resonant volume to be imaged. Magnetic response signals are detected by pick-up coils. In this
way, the tissue-specific differences of the relaxation time T1 and/or T2 may be used for
construction of the T1 and T2 weighted images showing optimal contrast of special tissue
features. In practice, for optimization of tissue contrast a variety of different pulse sequences
(e.g., the widely applied spin-echo methods) may be used (60)(61).
Generally, a magnetic resonance scanner is essentially defined by three hardware groups and
their parameters: a) the main magnet with its homogeneity over imaging volume; b) the
magnetic field gradient system with its linearity over the imaging volume; and c) the
radiofrequency (RF) system with its RF signal homogeneity and signal sensitivity over the
imaging volume. Whole-body MRI imposes very special demands on these system components
(62)(63):
a) The main magnet. Magnetic resonance imaging requires a very strong magnetic field
that has precisely the same magnitude and direction everywhere in the region we want to
image. One of the key properties used to describe the quality of a MRI system is the
uniformity, or homogeneity, of the applied magnetic field. For example, high-quality MRI
systems made for clinical use in hospitals will have magnetic fields that vary less than 5 parts
per million (ppm) over a 40 cm diameter spherical volume in the region desired for imaging.
b) The magnetic field gradient. As mentioned earlier, a key property of the static
magnetic field of a MRI system is its homogeneity, but anything we place inside the magnetic
field tends to change the magnetic field slightly. To make the magnetic field as uniform as
possible and to compensate for changes caused by different objects in the field, we shim the
field. Shimming is typically handled by placing small amounts of iron at specific locations within
long trays that line the cylindrical magnetic field coil; or by several set of wire coils. Once the
shimming is complete, the magnetic field is highly uniform over a central region where the
imaging takes place.
81

c) The radio frequency system. Radio frequency systems suitable for MRI are
characterized by RF excitation with homogeneous signals over an examination volume as large
as possible. Here, it is important that the excitation flip angle remains as constant as feasible,
as the image contrast is fundamentally influenced by this parameter.
Figure 2.34. A modern 9.4 Tesla MRI system (Bruker Biospin USR 94/20 instrument).
2.12.- Laboratory equipment.
Wilhelmy Plate method. Surface tensions () were determined by the Wilhelmy plate method
using a Krüss K12 surface tension equipment, equipped with a processor to acquire the data
automatically. The equipment was connected to a circulating water bath to keep the
temperature constant to within 0.01 0 C.
Drop tensiometer. Surface tension measurements were also carried out in a Track tensiometer
equipment (I.T. Concept, France) adapted to determine surface tension values in real time with
an accuaracy of 0.1 mN/m. Interfacial tension and interfacial rheology estimations are based
82

on the digital profile of a drop image and the resolution of the Gauss-Laplace equation. Win
Drop software (I.T. Concept, France) was used to obtain the surface tension values by means of
the axisymetric drop shape analysis. For air-water interface, an air-bubble upward was formed
at the tip of a U-shaped stainless steel needle (0.5 mm inner diameter) immersed in water bulk
phase. On the contrary, chloroform bubble downward was formed at the tip of a straight
stainless steel needle (0.5 mm inner diameter).
Langmuir-Blodgett trough. Surface-pressure isotherms were recorded in a Langmuir-Blodgett
(LB) Teflon trough (model 611 from Nima Technologies, Ltc., Coventry, U.K.).
Atomic force microscopy. The AFM measurements were performed in a JEOl instruments
(model JSPM-4210) in non-contact mode using nitride cantilevers NSC 15 from Micro Masch,
USA (typical working frequency and spring constant of 325 kHz and 40 N/m, respectively).
83

Laser light scattering. Dynamic and static light scattering (DLS and (SLS) intensities were
measured by means of an ALV-5000F (ALV-GmbH) instrument with vertically polarized incident
light of wavelength λ= 488 nm supplied by a CW diode pumped Nd;YAG solid-state laser
supplied by Coherent, Inc. and operated at 2 W.
Polarized optical microscopy. POM experiments were made by using a Zeiss Axioplan optical
microscope (Carl Zeiss Ltd., Welwyn Garden City, U.K.) A total magnification of either 50x or
100 X was used. A polarizer and analyzer were put in fixed position, orthogonal to one another
for the cross-polarizer imaging. Images were taken using a Kodak digital camera mounted on
the top of the microscope.
84

Laser confocal microscopy. Confocal microscopy was performed on a Leica TCS SP2 confocal
system mounted on a Leica DM-IRE2 upright microscope, using a 40X objective. For
fluorescence measurements, Thioflavin T was added to spherulitic samples and excited using
the 458 nm line of an argon ion laser. Simultaneously, transmission images were obtained with
crossed polarizers in place using the 633 nm line of a He-Ne laser.
Rheometry. The rheological properties of the samples were determined using a Bohlin CS10
rheometer with water bath temperature control. Coutte geometry (bob, 24.5 mm in diameter,
27 mm in height, cup, 26.5 in diameter, 29 mm in height) was used for fluid samples. Samples
of very high modulus were investigated using cone and plate geometry (diameter 40 mm,
angle 4 0 ). Frequency scans of storage (G’) and lost (G’’) moduli were recorded for selected
copolymer concentrations and temperatures with the instrument in oscillatory shear mode
and with the strain amplitude (A) maintained at a low value (A < 0.5 %) by means of auto-
stress facility of the Bohlin software. This ensured that measurements of G’ and G’’ were in the
linear viscoelastic region.
85

Transmission electron microscopy. TEM micrographs were acquired by using a Phillips CM-12
electron microscope operating at an accelerating voltage of 120 kV.
High resolution transmission electron microscopy and selected area electron diffraction. HR-
TEM images and SAED patterns were obtained with a transmission electron microscope (Carl-
Zeiss Libra 200 FE-EFTEM) operating at 200 kV.
Scanning electron microscopy. SEM micrographs were acquired by using a LEO-435 VP
scanning electron microscope (Leica Microsystems Gmb H, Wetlar, Germany) operating at an
accelerating voltage of 30 kV. Microanalysis of the scanning electron microscopy samples was
performed.
86

Fluorescence spectroscopy. Fluorescence was measured in a Cary Eclipse fluorescence
spectrophotometer equipped with a temperature control device and a multicell sample holder
(Varian instrument Inc.).
Ultraviolet-visible spectroscopy. UV-vis data were acquired by using a spectrophotometer DU
series 640, Beckman Coulter, (Fullerton, CA) operating at 190-110 nm.
Far and near-ultraviolet circular dichroism spectroscopy. CD spectra were obtained using a
JASCO-715 automatic recording spectropolarimeter (Jasco, Tokyo, Japan) with a JASCO PTC-
343 Peltier-type thermostated cell holder. Quartz cuvettes with 0.2cm pathlength were used.
CD spectra were recorded between 195 and 300 nm at 25 0 C.
87

Fourier transform infrared spectroscopy. An FTIR spectrometer (model IFS-66V; Bruker)
equipped with a horizontal ZnS ATR accessory. The spectra were obtained at a resolution of 2
cm -1 and 200 scans were accumulated to obtain a reasonable signal/noise ratio.
X-ray diffraction. XRD experiments were carried out using a Siemens D5005 rotating anode X-
ray generator. Twin Göbel mirror were used to produce a well-collimated beam of Cu Kα
radiation (λ = 1.5418 Å). XRD patterns were recorded with an imaging plate detector AXS F.Nr.
J2-394.
88

Superconducting quantum interference device. Magnetic susceptibility measurements were
carried out with a SQUID magnetometer (Quantum Design MPMS5, San Diego, CA).
Magnetic resonance measurements. Transverse and longitudinal relaxation times were
measured at 9.4 T (400 MHz) with a Brucker Biospin USR 94/20 instrument (Ettlingen,
Germany).
89

Contents
3.1
3.2
3.3
3.4
3.5
3.1.- Introduction
Introduction
Behaviour of block copolymers in solution
CHAPTER 3
Block copolymers
Mechanisms of morphologic control and thermodynamic
stability
Block copolymers of polyoxyalkylenes in aqueous solution
E12S10, E10S10E10 and E137S18E137 block copolymers
Block copolymers are a particular class of polymers that are included in a wide family of
nanomaterials, known as soft materials. Independently of the synthetic procedure, block
copolymers can simply be considered as being formed by two or more chemically
homogeneous polymer fragments joined together by covalent bonds, which can be found in
different positions along the polymeric chain (Figure 3.01) (64)(65). According to the number
of the building units, these polymers can be denominated as diblock, triblock and multiblock
copolymers. The molecular architecture of block copolymers can be as simple as a linear chain
or more complex like the molecular architecture of star or graft copolymers, which can be
controlled by means of the chemical synthetic procedures (9)(66)-(68).
Methodologies used in the synthesis of block copolymers allow designing any polymeric chain
with specific properties and architectures. Generally, the structure of a block copolymer is
composed of incompatible joint homopolymers. When a copolymer is molten, and
subsequently left cool down slowly, the incompatibilities between constituent building blocks
give rise to microphase separation, leading to the formation of ordered nanodomains. The
covalent linkage between the different polymeric chains prevents the separation in
macrodomains. The generation of these structural nanodomains is managed by the self-
assembling process of homologous segments that compose the block copolymer. Also, such
dimensional nanostructures depend on the volume fraction of blocks copolymer. Two
competing effects govern the thermodynamics of block copolymer melts. At high
91
93
95
96
97
91

temperatures, the chains are mixed homogeneously, as in any polymer melt. As the
temperature is reduced the tendency for the blocks to segregate is enhanced, i.e. the enthalpic
process of demixing is favoured. However, this is necessary accompanied by a reduction in the
entropy as the chain configuration becomes more constrained. The extent of segregation of
the copolymer may be expressed using the reduced parameter N. Here, is the Flory-Huggins
interaction parameter, which contains a significant enthalpic contribution, and is governed by
the incompatibility of the monomers; and N is the copolymer degree of polymerization,
reflecting the N-dependent translational and configurational entropy. The transition from
homogeneous melt of chains to heterogeneous melt of ordered microphase-separated
domains occurs at a critical value of N, depending on the composition of the copolymer (65).
In bulk, the minority block is segregated from the majority block forming regularly shaped and
uniformly-spaced nanodomains. The shape of the segregated domains in a diblock is governed
by the volume fraction of the minority block, f, and block incompability. Figure 3.1 shows the
equilibrium morphologies documented for diblock copolymers as an example. At a volume
fraction of ≈ 20 %, the minority block forms a body-centred cubic spherical phase in the matrix
of the majority block. It changes to hexagonally packed cylinders at a volume fraction ≈ 30 %.
Alternating lamellae are formed at approximately equal volume fractions for the two blocks. At
a volume fraction of ≈ 38 %, the minority block forms bicontinuous structures at moderate and
high incompatibility, respectively (64)(65)(69).
A Block B Block
Sphere Cylindric Bicontinuous Lamellar
Figure 3.1. Regular structures in block copolymer leaded by theirs self-assembling.
The physical properties of copolymeric materials have been applied in a wide range of fields.
For example, by decades copolymeric materials in solid state have been used as thermoplastic
and elastomeric materials, foams, and glues owing to their high compatibility, sensitivity, and
92

esistance to external physical factors (65). On other hand, the auto-association properties of
block copolymers in selective media allow the formation of structure-ordered aggregates, and,
for that reason, this type of polymeric materials has been received special attention in last
years for their used in high-nanotechnology applications, as shown below (8)(9).
3.2.- Behaviour of block copolymers in solution.
The hydrophilic and hydrophobic nature of amphiphilic block copolymer molecules lead to
their self-assembly into a variety of structures in solution. There are two basic processes that
characterize the phase behaviour of block copolymers in solution: micellization and gelation.
Micellization occurs in dilute solution of block copolymers in a selective solvent at a fixed
temperature above the cmc, as commented in more detail below. At higher copolymer
concentrations, the micelles can order onto a lattice above a critical gel concentration (cgc).
These regimes are illustrated in Figure 3.2.
Figure 3.2 Illustration of cmc and cgc in ablock copolymer solution.
When a block copolymer is dissolved in a selective solvent, that is, a solvent which is good for
one of the blocks and bad for the other one, the lyophobic part of the macromolecule tends to
segregate and auto-associate with their neighbouring molecules, leading to the formation of
stable aggregates with specific structures, in order to avoid the direct contact between solvent
molecules with the insoluble copolymer block (70). The spontaneous aggregation of block
copolymers is carried out either when the concentration of the macromolecule is near to their
critical micellar concentration, or on the other hand, by changes in the solution temperature.
In this case, the critical micellar temperature is reached. Both parameters are fundamental to
characterize the association properties of a copolymer-solvent system (71). Despite the cmc
does not correspond to a thermodynamic property of the system, it can simply be defined
phenomenollogically as the concentration at which a sufficient number of micelles is formed to
93

e detected by a given method. Thermodynamically, for copolymers with a narrow block-
length, the association process corresponds to equilibrium between molecules (unimers), A,
and micelles, AN, containing N molecules. This can be written as:
If the association number is large and independent of the temperature then and
the standart Gibbs energy of association is:
Under this condition, for molecules and micelles in equilibrium just above the cmc
Figure 3.3. Schematic representation of block copolymer micelles in aqueous solution, dense
hydrophobic core is constituted by the hydrophobic block, while the micellar shell is formed by
the hydrophilic block.
The micellization process of amphiphilic block copolymers has been studied in a wide range of
selective solvents: from water to organic polar solvents and non-polar solvents, as well as
ecological solvents like supercritic fluids and ionic liquids (72). The simplest structural
aggregates formed by block copolymer are called micelles, by the analogy that exists with the
structure of common micelle formed by the convectional surfactants in aqueous media (Figure
3.3) (73).
3.1
3.2
94

In aqueous media, the micelle structure consists of a dense hydrophobic core surrounded by a
hydrophilic shell which is exposed toward the bulk solvent phase in a spherical configuration;
the micelles can change their size from 5 to some hundreds nanometers and can possess
different geometries (spheres, worn-like, and so on) (74)-(78).
The various reported micellar-like morphologies are primarily a result of the inherent
molecular curvature and how this influences the packing of the copolymer chains: specific self-
assembled nanostructures can be targeted according to a dimensionless packing parameter, ,
which is defined as
, where is the volume of the hydrophobic chains, is the
optimal area of the head group, and, is the legth of the hydrophobic tail, as occurred for
classical surfactants. Therefore, the packing parameter of a given molecule usually dictates its
most likely self-assembled morphology. As a general rule, spherical polymeric micelles are
favored when
, cylindrical micelles when
structures (vesicles, also known as polymersomes) when
, and enclosed membrane
Despite the structured aggregates from block copolymers formed in aqueous solution are
similar to those produced by the self-assembly of small surfactant molecules such as lipids and
detergents, the polymeric nature of the amphiphilic macromolecules provides new special
properties to these structural complexes, as stability and functionality. In this way, their
potential applications in different technological areas are largely increasing, including
materials science, bioengineering, medicine, pharmaceutics industry, and ink and paint
industry (70)(79).
3.3.- Mechanism of morphologic control and thermodynamic stability.
The conformation of the block copolymer chains depends on the interaction between chain
segments and solvent molecules. In aqueous solution, the structure of the aggregates is
governed by the balance of the interaction forces between the block copolymer molecules
with water molecules, and by the intrachain interactions between the blocks which constitute
the copolymer molecule (where the hydrophilic chain expands, avoiding unfavourable
segment-segment interactions and the hydrophobic part contract to minimize the contact with
solvent molecules).
It is possible to achieve a precise control of the size and shape of polymeric aggregates, and
.
95

subsequently, of their thermodynamic stability by modification of some factors such as block
copolymer molecular weight, the relatively length of both hydrophobic and hydrophilic blocks,
the chemical nature of the different monomer units and the architecture of the polymeric
backbone. Firstly, this can be achieved by means of the block copolymer synthetic processes.
However, the synthesis of block copolymers is time-consuming and expensive, disadvantages
that have to be taken into account. A simpler alternative is the possibility of manipulating the
block copolymer solution properties through the change of external parameters such as
temperature, salinity, pH, addition of cosolvents… Under this context, it is possible to obtain
different aggregate sizes or morphologies using the same block copolymer or the same family
of copolymers. The morphology of the structured aggregates can be determined by a balance
of different factors such as the solvent properties, the conformation of the hydrophobic chains
into the micelle core, the repulsion between hydrophilic blocks into the micelle shell, and the
surface tension between the micelle cores and the bulk solution (77)(78).
3.4.- Block copolymers of polyoxyalkylenes in aqueous solution.
The block copolymers of polyoxyalkylenes are composed of a hydrophilic block constituted by
poly(ethylene oxide) (OCH2CH2 ethylene oxide unit, E) and by a second hydrophobic block that
can be made of, for example, poly(propylene oxide) (P), poly(styrene oxide) (S), or
poly(butylene oxide) (B), amongst others. In aqueous medium, polyoxyalkylene amphiphilic
block copolymer contains both the hydrophilic and hydrophobic blocks that can associate to
usually form spherical micelles in dilute aqueous solution. Increasing the concentration leads
to the formation of lyotropic crystal mesophases (gels) where the structural objects are
micelles, usually efficiently packed in a body-centered or face-centered cubic structure as a
result of their interaction as hard spheres (80)(81).
For poly(oxyalkylenes) block copolymers, values of the cmc are affected by the variation of
hydrophilic polyoxyethylene and hydrophobic block lengths. It is known that the dependence
of the cmc on the number of E units in a copolymer is small unless the hydrophobic-block
length is short. Indeed, when comparing results for two copolymers of nominally the same
hydrophobic-block length, the effect of E-block length may well be less than the variation
caused by the uncertainty in hydrophobic-block length. However, within a series of copolymers
it is desirable to account for variation in E-block length. Taking as an example PE-PP-PE
Pluronic block copolymers, micelle formation of this class of polymers in water is believed to
96

esult from dehydratation of the hydrophobic block with increasing temperature, water being
a selective solvent for PE. This explains why the solution properties of Pluronic polymers are
strongly temperature-dependent. For most PE-PP-PE copolymers, the cmt increases with
decreasing in copolymer concentration. The number of oxypropylene units has a strong effect
on the micellization, copolymers with large hydrophobic PP block length forming micelles at
much lower concentrations. Indeed, the cmc is found to decrease exponentially with the PP
block length. On the other hand, the PE block has a smaller influence on the micellization. An
increase in the number of PE units leads to small increases in the cmc and cmt values. The cmt
and cmc decrease with increasing the total copolymer molecular weight, if comparisons are
made for a constant PP/PE ratio. On the other hand, the large positive enthalpy of
micellization, and the standard free energy of micellization observed on the self-assembly of
PE-PP-PE demonstrate that the association process is entropy driven. Chain architecture has
also a great influence on micelle formation. For example, the tendency for micellization of PP-
PE-PP copolymer is reduced compared to a PE-PP-PE copolymer of the same composition.
3.5.- E12S10, E10S10E10 and E137S18E137 block copolymers
Our research group has been collaborating during last years with research groups of the School
of Pharmacy and Pharmaceutical Sciences and the School of Chemistry of the University of
Manchester in synthesizing block copolymers formed by ethylene oxide and styrene oxide
blocks (81)-(83). In this regard, it has been previously demonstrated that the micellization
process of diblock copolymers of this type has not cmt values. Moreover, they usually have
small cmc values and micellization enthalpies (micH) as a consequence of the high
hydrophobicity of the styrene oxide block. Triblock copolymers show cmc values higher than
diblock ones for the same hydrophobic block length. On the other hand, the cmc values of the
triblocks weakly depend of the hydrophilic block length.
Block copolymers studied in this work were: E12S10, E10S10E10 and E137S18E137, (where E
represents the oxyethylene unit, S represents the oxystyrene unit, and the subscripts
correspond to block lengths). These copolymers were synthesised by sequential anionic
polymerisation of the two monomers, oxyethylene and oxystyrene. Oxyrane compounds are
heterocyclic compounds constructed by two carbon atoms and one oxygen atom into the
cyclic structure (Figure 3.4) (84).
97

a) O
b)
H 2C CH 2
O
H 2C CH
Figure 3.4. Oxyranes used in the synthesis of the block copolymers a) ethylene oxide and b)
styrene oxide.
Anionic polymerization proceeds via organometallic sites, carbanions (or oxanions) with
metallic counterions. Carbanions are nucleophiles; consequently, the monomers that can be
polymerized by anionic polymerization are those bearing an electro-attractive substituent on
the polymerizable double bound. Initiation of polymerization is accomplished by analogous
low molecular weight organometallic compounds (initiators). A wide variety of initiators has
been used so far in order to produce living polymers. Among them, the most widely used are
organolithiums. The main requirement for employment of an organometallic compound as an
anionic initiator is its rapid reaction with the monomer at the initiation step of the
polymerization reaction and, specifically, with a reaction rate larger than that of the
propagation step. This leads to the formation of polymers with narrow molecular weight
distributions because all active sites start polymerizing the monomer almost at the same time.
Propagation proceeds through nucleophilic attack of the carbanionic site onto a monomer
molecule with reformation of the first anionic active center. The situation is similar in the case
of the ring opening polymerization of cyclic monomers containing heteroatoms (oxyranes,
lactones, siloxanes, etc.). Under appropriate experimental conditions, due to the absence of
termination and chain transfer reactions, carbanions (or, in general, anionic sites) remain
active after complete consumption of monomers, giving the possibility of block copolymer
formation, in the simplest case, by introduction of a second monomer into the polymerization
mixture. However, a variety of different synthetic strategies have been reported for the
preparation of linear block copolymers by anionic polymerization.
As an example, in the polymerisation reaction of a monofunctional poly(oxyethylene) the
addition of ethylene oxide (E) is initiated by a monofunctional alcohol (ROH), part of which is in
the form of its alkali-metal salt (e.g., RO-K + .) Initiation is instantaneous, and the subsequent
reaction scheme is written:
98

where Em represents a growing chain, the active centre is an ion pair, and the rapid
equilibration ensures that all chains are equally likely to grow. This ideal scheme would
produce polymers with Mn= (mass of E polymerised)/(moles of initiator used) and a Poisson
distribution of chain lengths. In practice, the chain length distribution can be widened by a
slow initiation. In the absence of termination, the system is living and is ideal for preparation
of block copolymers. The same scheme applies to styrene oxide.
It is known that block poly(oxyalkylene) copolymers with hydrophobic blocks formed by
propylene oxide or butylene oxide (either diblock or triblock architectures) have a
spontaneous formation of spherical and cylindrical micelles in dilute aqueous solution. On the
other hand, formation of vesicular structures for poly(oxyalkylene) block copolymers needs
oftenly an energy supply via mechanical, sonochemical or electrical methods (85)(86).
Nevertheless, it has been previously reported the spontaneous formation of vesicles (87)(88),
where these types of structures are likely to form because of relatively large volume to length
ratios of the hydrophobic block and the relative lengths of the poly(oxyethylene) blocks. In the
case of block copolymers containing poly(oxystyrene) blocks as the hydrophobic block, the
formation of elongated micelles has been unusual, whereas vesicle formation has not been yet
reported.
99

Contents
4.1
4.2
4.3
4.3.1
CHAPTER 4
Papers on block copolymers
Self-Assembly process of different poly(oxystyrene)-
poly(oxyethylene) block copolymers: spontaneous
formation of vesicular structures and elongated micelles.
Surface properties of monolayers of amphiphilic
poly(ethylene oxide)-poly(styrene oxide) block copolymers.
Appended papers
Relevant aspects
97
107
117
118

Self-Assembly Process of Different Poly(oxystyrene)-Poly(oxyethylene)
Block Copolymers: Spontaneous Formation of Vesicular Structures
and Elongated Micelles
Josué Juárez, † Pablo Taboada,* ,† Miguel A. Valdez, ‡ and Víctor Mosquera †
Departamento de InVestigación en Polímeros y Materiales y Departamento de Física, UniVersidad de
Sonora, Sorales Resales y TransVersal, 83000 Hermosillo Sonora, Mexico, and Laboratorio de Física de
Coloides y Polímeros, Grupo de Sistemas Complejos, Departamento de Física de la Materia Condensada,
Facultad de Física, UniVersidad de Santiago de Compostela, Spain
ReceiVed February 12, 2008. ReVised Manuscript ReceiVed March 26, 2008
In the present work, we investigated the micellization, gelation, and structure of the aggregates of three poly(ethylene
oxide)-polystyrene oxide block copolymers (E12S10, E10S10E10, and E137S18E137, where E denotes ethylene oxide and
S styrene oxide and the subscripts the block length) in solution. Two of them have similar block lengths but different
structures (E12S10 and E10S10E10) and the other has longer blocks (E137S18E137). For the first time, the spontaneous
formation of vesicles by a poly(oxystyrene)-poly(oxyethylene) block copolymer is reported. These vesicular structures
are present when copolymer E12S10 self-assembles in aqueous solution in coexistence with spherical micelles, as
confirmed by the size distribution obtained by dynamic light scattering and pictures obtained by polarized optical
microscopy, and transmission and cryo-scanning electron microscopies. Vesicle sizes vary between 60 and 500 nm.
On the other hand, for copolymers E10S10E10 and E137S18E137, only one species is found in solution, which is assigned
to elongated and spherical micelles, respectively. If we compare the high aggregation number derived by static light
scattering for the triblock block copolymer micelles, with the maximum theoretical micellar dimensions compatible
with a spherical geometry, we can see that the micellar geometry cannot be spherical but must be elongated. This
is corroborated by transmission electron microscopy images. On the other hand, tube inversion was used to define
the mobile-immobile (soft-hard gel) phase boundaries. To refine the phase diagram and observe the existence of
additional phases, rheological measurements of copolymer E137S18E137 were done. The results are in good agreement
with previous values published for other polystyrene oxide-poly(ethylene oxide) block copolymers. In contrast, copolymers
E12S10 and E10S10E10 did not gel in the concentration range analyzed. Thus, only certain concentrations of copolymer
E10S10E10 were analyzed by rheometry, for which an upturn in the low-frequency range of the stress moduli was
observed, denoting an evidence of an emerging slow process, which we assign to the first stages of formation of an
elastic network.
Introduction
Block copolymers of hydrophilic poly(oxyethylene)
(OCH2CH2, E) oxyethylene unit) and hydrophobic poly(oxystyrene)
(OCH2CH(C6H5), S ) oxyphenylethylene unit) are
denoted as either EmSn,SnEmor EmSnEm,SnEmSn, depending on
the sequence of polymerization or architecture, respectively. They
associate to usually form spherical micelles in dilute aqueous
solution. 1,2 Increases in concentration lead to the formation of
lyotropic crystal mesophases (gels) where the structural objects
are the micelles usually efficiently packed in a body-centered or
face-centered cubic structure as a result of being effectively
interacting as hard spheres. 3
Aggregate morphology is determined primarily by a force
balance among three contributions: the core-chain stretching,
corona-chain repulsion, and interfacial tension between the core
and the outside solution. Consequently, factors that influence the
above balance can be used to control the aggregate architecture:
* Author to whom correspondence should be addressed. Telephone:
0034981563100, ext.: 14042. Fax: 0034981520676. E-mail: fmpablo@usc.es.
† Universidad de Santiago de Compostela.
‡ Universidad de Sonora.
(1) Crothers, M.; Attwood, D.; Collett, J. H.; Yang, Z.; Booth, C.; Taboada,
P.; Mosquera, V.; Ricardo, N. P. S.; Martini, L. G. A. Langmuir 2002, 18, 8685–
8691.
(2) Yang, Z.; Crothers, M.; Ricardo, N. M. P. S.; Chaibundit, C.; Taboada,
P.; Mosquera, V.; Kelarakis, A.; Havredaki, V.; Martini, L.; Valder, C.; Collett,
J. H.; Attwood, D.; Heatley, F.; Booth, C. Langmuir 2003, 19, 943–950.
(3) Castelletto, V.; Hamley, I. W.; Crothers, M.; Attwood, D.; Yang, Z.; Booth,
C. J. Macromol. Sci., Phys. 2004, 43, 13–27.
Langmuir 2008, 24, 7107-7116
10.1021/la8004568 CCC: $40.75 © 2008 American Chemical Society
Published on Web 06/12/2008
7107
polymer molecular weight, relative block lengths, hydrophobic
block structure, solution conditions, and so forth. For a given
value of the association number (Nw) for a poly(oxyalkylene)
block copolymer, the average volume of a micelle core is readily
estimated, and the corresponding spherical radius (rc) can be
compared with the length l (or half-length, l/2, in the case of
triblock copolymers) of the extended hydrophobic blocks. With
allowance made for a Poisson distribution of hydrophobic blocks,
experience shows that spherical (or near-spherical) micelles will
be formed if l (l/2 for triblock architecture) is approximately
equal or greater than the value of rc. 4,5 If this does not occur,
other types of assemblies are observed under certain solution
conditions. For example, a cylindrical geometry is expected as
the solution temperature is increased because of an increase in
the aggregation number derived from block copolymer dehydration:
6 the core blocks are stretched as the radius of a spherical
micelle core is increased eventually to reach a point at which the
enthalpy penalty forces the transition to a cylindrical geometry.
Thus, formation of cylindrical micelles has been widely studied
in block poly(oxyalkylene) copolymers with hydrophobic blocks
(4) Taboada, P.; Velazquez, G.; Barbosa, S.; Castelletto, V.; Nixon, S. K.;
Yang, Z.; Heatley, F.; Hamley, I. W.; Ashford, M.; Mosquera, V.; Attwood, D.;
Booth, C. Langmuir 2005, 21, 5263–5271.
(5) Ricardo, N. M. P. S.; Pinho, M. E. N.; Yang, Z.; Attwood, D.; Booth, C.
Int. J. Pharm. 2005, 300, 22–31.
(6) Booth, C.; Attwood, D. Macromol. Rapid Commun. 2000, 21, 501–527.
103

7108 Langmuir, Vol. 24, No. 14, 2008 Juárez et al.
formed by propylene oxide 6–9 and butylene oxide 10–13 with
diblock and triblock architectures. On the other hand, formation
of vesicular structures for poly(oxyalkylene) block copolymers
has often needed energy supply via mechanical, sonochemical,
or electrical methods. 14,15 Nevertheless, some works have reported
the spontaneous formation of vesicles, denoting that these types
of structure are likely to form because of relatively large volumeto-length
ratios of the hydrophobic block and the relative lengths
of the poly(oxyethylene) blocks. 16–18
In the case of block copolymers containing poly(oxystyrene)
blocks, the formation of elongated micelles has been unusual,
whereas vesicle formation has not been reported. Probably, neither
the composition nor the solvent conditions of the analyzed
poly(ethylene oxide)-polystyrene oxide block copolymers promote
the clear formation of other aggregate structures than
spherical micelles. Only Yang et al. 19 have compared the
association number (Nw) of copolymer micelles of copolymers
E17B8 and E17S8 and found that substitution of the B block by
the S block brought about a large rise in Nw, which was larger
than the theoretical increase expected for spherical micelles.
They hypothesized that the difference in Nw between these two
copolymers might be explained in terms of micelle elongation,
but no direct measurements were done. Moreover, in a recent
report Elsabahy et al. 20 studied the drug solubilization capacity
and release kinetics in vitro of the anticancer drug docetaxel by
poly(oxystyrene)-poly(oxyethylene) block copolymers. These
authors have demonstrated the existence of an elongated structure
of micelles formed by copolymers E45S15 and E45S26 by TEM.
Nevertheless, these authors also pointed out the possible influence
of residual styrene oxide homopolymer from the synthesis step
dissolved in the micelle core to elongate the aggregate structure,
as discussed earlier by Nagarajan. 21
Thus, in the present work, we studied the self-assembly
properties and the different resulting aggregate structures of three
copolymers with poly(oxyethylene) and poly(oxystyrene) blocks
(E12S10,E10S10E10, and E137S18E137), one diblock and two triblock
copolymers, respectively, with very different volume-to-length
ratios of the hydrophobic block and relative lengths of the
poly(oxyethylene) blocks, which should help in controlling
aggregate morphology. The study was conducted by using
different techniques such as surface tension, light scattering,
transmission and scanning electron microscopies, polarized optical
microscopy, and rheology. In fact, for copolymer E12S10
(7) Chu, B.; Zhou, Z. Physical Chemistry of Polyoxyalkylene Block Copolymer
Surfactants. In Nonionic Surfactants: Polyoxyalkylene Block Copolymers; Nace,
V. M., Ed.; Surfactant Science Series 60; Marcel Dekker: New York, 1996.
(8) Hvidt, S.; Jørgensen, E. B.; Brown, W.; Schillén, K. J. Phys. Chem. 1994,
98, 12320–12328.
(9) Gangaly, R.; Awal, V. K.; Hassan, P. A. J. Colloid Interface Sci. 2007,
325, 693–700.
(10) Kelarakis, A.; Havredaki, V.; Booth, C.; Nace, V. M. Macromolecules
2002, 35, 5591–5594.
(11) Chaibundit, C.; Ricardo, N. M. P. S.; Crothers, M.; Booth, C. Langmuir
2002, 18, 4277–4283.
(12) Li, H.; Yu, G.-E.; Price, C.; Booth, C.; Fairclough, J. P. A.; Ryan, A. J.;
Mortensen, K. Langmuir 2003, 19, 1075–1081.
(13) Chaibundit, C.; Sumnatrakool, P.; Chinchew, S.; Kanatharana, P.;
Tattershall, C. E.; Booth, C.; Yuan, X.-F. J. Colloid Interface Sci. 2005, 283,
544–554.
(14) Battaglia, G.; Ryan, A. J. J. Am. Chem. Soc. 2005, 127, 8757–8764.
(15) Zipfel, J.; Lindner, M.; Tsinou, M.; Alexandridis, P.; Richtering, W.
Langmuir 1999, 15, 2599–2602.
(16) Battaglia, G.; Ryan, A. J. Nature Mat. 2005, 4, 869–876.
(17) Bryskhe, K.; Jansson, J.; Topgaard, D.; Schillén, K.; Olsson, U. J. Phys.
Chem. B 2004, 108, 9710–9719.
(18) Harris, J. K.; Rose, G. D.; Bruening, M. L. Langmuir 2002, 18, 5337–
5342.
(19) Yang, Z.; Crothers, M.; Attwood, D.; Collet, J. H.; Ricardo, N. P. S.;
Martini, L. G. A.; Booth, C. J. Colloid Interface Sci. 2003, 263, 312–317.
(20) Elsabahy, M.; Perron, M.-E.; Bertrand, N.; Yu, G.-E.; Leroux, J.-C.
Biomacromolecules 2007, 8, 2250–2257.
(21) Nagarajan, R. Colloids Surf., B 1999, 16, 55–72.
Table 1. Molecular Characteristics of the Copolymer a
Mn/g mol -1
(NMR)
wt%S
(NMR)
Mw/Mn
(GPC) Mw/g mol -1
coexistence of spherical micelles with vesicular structures is
detected. To the best of our knowledge, the spontaneous formation
of vesicles by a poly(oxystyrene)-poly(oxyethylene) block
copolymers is reported for the first time. On the other hand,
formation of elongated micelles upon self-assembly of copolymer
E10S10E10 in dilute solution is elucidated from light scattering
and transmission electron techniques. In the case of copolymer
E137S18E137, typical spherical micelles are observed as expected.
The micellization properties of the latter copolymer had been
already partially studied previously, and the present study
completes the gap. 22,23 Finally, comparison of the self-assembly
properties of the present copolymers with structurally related
poly(ethylene oxide)-propylene oxide and poly(ethylene oxide)butylene
oxide block copolymers is also made.
Experimental Section
Materials. The synthesis of the copolymers was described
previously in detail. 1,2 Table 1 shows the molecular characteristics
of the copolymers. Water was double distilled and degassed before
use.
Clouding. Solutions in small tubes (10 mm in diameter) were
prepared by weighting copolymer and mixing with water in the
mobile state before being stored at low temperature for several days
(T ≈ 5 °C). Tubes containing 0.5 cm3 of solution were immersed
in a water bath, which was heated at a heating/cooling rate of 0.2
°C min-1 E12S10 1660 73 1.05 1760
E10S10E10 1980 55 1.06 2130
E137S18E137 14200 15 1.06 15100
a
Estimated uncertainty: Mn to (3%;wt%Sto(1%, Mw/Mn to (0.01.
Mw calculated from Mn and Mw/Mn.
from5to90°C. Clouding was detected by the eye. The
tubes were also inverted to check the possibility of gel formation.
The change from a mobile to an immobile system (or vice versa)
was determined by inverting the tube at 1-min intervals.
Surface Tension Measurements. Surface tensions (γ) ofthe
copolymers were measured by the Wilhelmy plate method using
Krüss K-12 surface tension equipment, equipped with a processor
to acquire the data automatically. The equipment was connected to
a circulating water bath to keep the temperature constant at the
corresponding temperature to within (0.01 °C. The plate was cleaned
by being washed with doubly distilled water followed by being
heated in an alcohol flame. A copolymer stock solution was prepared
with distilled water and diluted as required. For measurements, a
solution was equilibrated at the proper temperature and the surface
tension was recorded at 15-min intervals until a constant value was
reached, a process that took 12-36 h depending on concentration.
The accuracy of measurement was checked by frequent determination
of the surface tension of pure water.
Light Scattering Measurements. Dynamic and static light
scattering (DLS and SLS) intensities were measured for copolymer
solutions at 20 and 50 °C for E137S18E137 by means of an ALV-
5000F (ALV-GmbH) instrument with vertically polarized incident
light of wavelength λ ) 532 nm supplied by a CW diode-pumped
Nd; YAG solid-state laser supplied by Coherent, Inc. and operated
at 400 mW. The intensity scale was calibrated against scattering
from toluene. Measurements were made at the scattering angle, θ,
of 90° to the incident beam. Solutions were equilibrated at each
chosen temperature for 30 min before we made a measurement.
Experiment duration was in the range 5-15 min, and each experiment
was repeated two or more times. To eliminate dust, samples were
filtered through Millipore Millex filters (Triton free, 0.45-0.8-µm
porosity).
(22) Ricardo, N. M. P. S.; Chaibundit, C.; Yang, Z.; Attwood, D.; Booth, C.
Langmuir 2006, 22, 1301–1306.
(23) Pinho, M. E. N.; Costa, F. M. L. L.; Filho, F. B. S.; Ricardo, N. M. P. S.;
Yeates, S. G.; Attwood, D.; Booth, C. Int. J. Pharm. 2007, 328, 95–98.
104

Self-Assembly of OCH2CH(C6H5)-OCH2CH2 Block Copolymers Langmuir, Vol. 24, No. 14, 2008 7109
The correlation functions from DLS were analyzed by the CONTIN
method to obtain intensity distributions of decay rates (Γ). 24 The
decay rate distributions gave distributions of apparent diffusion
coefficient (Dapp ) Γ/q 2 , q ) (4πns/λ) sin(θ/2), where ns ) refractive
index of solvent), and integrating over the intensity distribution
gave the intensity-weighted average of Dapp. Values of the apparent
hydrodynamic radius (rh,app, radius of hydrodynamically equivalent
hard sphere corresponding to Dapp) were calculated from the
Stokes-Einstein equation
r h,app ) kT/(6πηD app)
where k is the Boltzmann constant and η is the viscosity of water
at temperature T.
The basis for analysis of SLS was the Debye equation
mic
K * c/(I - Is ) ) 1⁄Mw + 2A2c + ... (2)
where I is intensity of light scattering from solution relative to that
from toluene, Is is the corresponding quantity for the solvent, c is
the concentration (in g dm-3 ), Mw mic is the mass-average molar mass
of the solute, A2 is the second virial coefficient (higher coefficients
being neglected), and K* is the appropriate optical constant which
includes the specific refractive index increment, dn/dc. Values of
the specific refractive index increment, dn/dc, and its temperature
increment were taken from previous reports. 1,2 The possible effect
of the different refractive indices of the blocks on the derived molar
masses of micelles has been found to be negligible. 25 Other quantities
used were the Raleigh ratio of toluene for vertically polarized light,
Rv ) 2.57 × 10-5 [1 + 3.68 × 10-3 (t - 25)] cm-1 (t in °C) and the
refractive index of toluene, n ) 1.4969[1-5.7 × 10-4 (t - 20)]. 26
Polarized Optical Microscopy (POM). POM experiments were
made by using a Leica TCS-SP2 microscope equipped with cross
polarizers in copolymer sample before being filtered. Samples after
filtration did not allow measurements of the typical lamellae patterns
because of insufficient instrument resolution.
Transmission Electron Microscopy (TEM). Samples for TEM
were prepared by evaporation of a 2.5 g dm-3 aqueous micellized
filtered copolymer solutions under air (with 0.8-µm pore filters). A
drop of copolymer solution was placed on an electron microscope
copper grid and stained with 2% (v/v) of phosphotungstic acid.
After drying, electron micrographs of the sample were recorded
with a Phillips CM-12 electron microscope.
Cryo-Scanning Electron Microscopy (Cryo-SEM). Cryo-
SEM micrographs were acquired by using a JEOL JSM-6360LV
microscope with accelerating voltage of 0.5-30 keV. Solutions were
filtered and flash frozen in liquid ethane and stored in liquid nitrogen.
They were transferred to a cryo preparation chamber at -90 °C and
held at this temperature for 2 min in a vacuum to sublime the surface
ice and sputter-coated with conductive chromium.
Rheometry. The rheological properties of the samples were
determined using a Bohlin CS10 rheometer with water bath
temperature control. Couette geometry (bob, 24.5 mm in diameter,
27 mm in height; cup, 26.5 mm in diameter, 29 mm in height) was
used, with 2.5 cm3 of sample being added to the cup in the mobile
state. Samples of very high modulus were investigated using coneand-plate
geometry (diameter 40 mm, angle 4°). A solvent trap
maintained a water-saturated atmosphere around the cells. Frequency
scans of storage (G′) and loss (G″) moduli were recorded for selected
copolymer concentrations and temperatures with the instrument in
oscillatory shear mode and with the strain amplitude (A) maintained
at a low value (A < 0.5%) by means of autostress facility of the
(24) Provencher, S. W. Makromol. Chem. 1979, 180, 201–209.
(25) Mai, S.-M.; Booth, C.; Kelarakis, A.; Havredaki, V.; Ryan, J. A. Langmuir
2000, 16, 1681–1688.
(26) (a) El-Kashef, H. ReV. Sci. Instrum. 1998, 69, 1243–1245. (b) Liu, T.;
Schuch, H.; Gerst, M.; Chu, B. Macromolecules 1999, 32, 6031–6042. (c) The
temperature dependence of the Raleigh ratio was not found in the literature and,
as a working approximation, values of Rv for toluene were adjusted relative to
those published for benzene. Gulari, E.; Chu, B.; Liu, T. Y. Biopolymers 1979,
18, 2943. The adjustment is small over the temperature range involved.
(1)
Figure 1. Temperatures at which turbidity was visually observed on
heating dilute solutions of copolymers (9)E12S10 and (O)E10S10E10. The
lines are to guide the eye.
Table 2. Critical Micelle Concentration (cmc), Surface Excess
Concentration (Γmax), Minimum Area per Molecule (Amin), Gibbs
°
Free Energy of Micelle Formation (∆Gmic),
and Gibbs Free
°
Energy of Adsorption (∆Gads)
of the Present Copolymers a
polymer T (°C) cmc (g dm -3 ) Γmax (10 -6 mol m -2 ) Amin (nm 2 )
E12S10 15 0.006 0.97 1.7
E10S10E10 15 0.012 0.91 1.8
E137S18E137 15 0.017 0.40 4.1
50 0.013 0.46 3.6
a
Estimated uncertainties: cmc, Γmax, Amin to (10%.
Bohlin software. This ensured that measurements of G′ and G″ were
in the linear viscoelastic region. Temperature scans were recorded
for selected copolymer concentrations at a frequency of 1 Hz. The
samples were heated at ca. 0.5 °C in the range 5-80 °C.
Measurements on solutions of low modulus (G″ ) 1-10 Pa) which
felt outside the range for satisfactory autostress feedback were
rejected.
Results
Clouding. Clouding temperatures obtained visually over a
wide concentration range are plotted in Figure 1. The overall
shape for copolymers E12S10 and E10S10E10 is as expected for
solutes with a lower critical solution temperature. Solutions of
copolymer E12S10 were only transparent at very low concentration,
and opacity increased in a narrow interval as the concentration
increased. Copolymer E10S10E10 solutions were clear in a wider
temperature and concentration range. Meanwhile, solutions of
copolymer E137S18E137 remained clear to the eye in the whole
temperature and concentration range analyzed. As described in
subsequent sections, the solutions were formed by copolymer
aggregates over the concentration range involved and the detailed
shape of the cloud point curve for the former copolymers has
to be discussed in that context. This reflects the different behavior
of the three copolymers as will be commented in detail below.
We return to this point after presentation of the light scattering
results. On the other hand, gelation was only observed for
copolymer E137S18E137 at concentrations larger than 15% (w/v)
with no lower temperature boundary.
Critical Micelle Concentration and Surface Properties of
Copolymers. Surface tensions of solutions of samples corresponding
to copolymers E137S18E137,E10S10E10, and E12S10 were
investigated, as set out in Table 2. Plots of surface tension against
log(concentration) are plotted in Figure 2. The concentration at
105

7110 Langmuir, Vol. 24, No. 14, 2008 Juárez et al.
Figure 2. Surface tension (γ) vs logarithm of concentration (g dm -3 )
of copolymers (O) E10S10E10 and (9) E12S10 at 15 °C and E137S18E137 at
(2) 15 and (3) 50°C in aqueous solution.
which the surface tension reached a steady value served to define
the cmc. Representative values of the cmc and of the surface
tension at the cmc (γcmc) are listed in Table 2. It can be observed
that the triblock copolymers have higher cmc values than the
diblock copolymer. The effect is originated by the entropy of
the triblock chains constrained by two block junctions in the
core-shell interface of the aggregate, compared with only one
constrain for the diblock chain. The present values for solutions
of copolymers E137S18E137, E10S10E10, and E12S10 at 15 °C are
in good agreement with previously reported values for other
diblock and triblock copolymers of ethylene oxide and styrene
oxide. 27,28
On the other hand, the surface excess concentration, Γmax, and
the minimum area per copolymer molecule, Αmin, at the air/
water interface were obtained using the surface tension measurements
and the following equations:
Γmax ) - 1
RT( ∂γ
∂ ln c) T,p
Amin ) 1/NAΓmax where R is the gas constant, NA is Avogadro’s number, and c is
the copolymer concentration.
Because of their higher hydrophobicity, copolymers E12S10 and
E10S10E10 display a larger interface coverage than E137S18E137, whose
molecules also need more space to be accommodated because of
the longer length. 28 On the other hand, for the triblock E137S18E137
there is a decrease in the value of A as the temperature rises as a
consequence of the increase in the thermal motions of the polymer
chains and the dehydration of the hydrophilic blocks. 29,30
(27) Booth, C.; Attwood, D.; Price, C. Phys. Chem. Chem. Phys. 2006, 8,
3612–3622.
(28) Barbosa, S.; Cheema, M. A.; Taboada, P.; Mosquera, V. J. Phys. Chem.
B 2007, 111, 10920–10928.
(29) Kelarakis, A.; Havredaki, V.; Yu, G.-E.; Derici, L.; Booth, C. Macromolecules
1998, 31, 944–946.
(3)
(4)
Figure 3. Intensity fraction distributions of the apparent hydrodynamic
radius for (a) copolymer E137S18E137 (60 g/dm -3 at 20 °C) and (b)
copolymer E12S10 (2.5 g/dm -3 at 10 °C).
Vesicular Formation in Copolymer E12S10. Intensity fraction
distributions of decay times for the three copolymers are
shown in Figure 3. In the case of copolymers E10S10E10 and
E137S18E137, the distribution contained one single peak (see Figure
3a for copolymer E137S18E137 as an example). In contrast, three
single narrow peaks were observed for copolymer E12S10 at
suitable concentrations to be measured by DLS (Figure 3b). These
peaks did not vary in a systematic way in the narrow copolymer
concentration range analyzed (0.5-3 gdm -3 ) provided that
solutions were not completely clear (turbidity increased as the
copolymer concentration rises), so a true hydrodynamic radius
could not be obtained (plot not shown). Because of the translucent
aspect of these solutions and to know if these solutions were
isotropic or not, we analyzed some of the samples by POM.
Surprisingly, the typical patterns displayed by spherical lamellae
were observed for all the samples in the concentration range
anlyzed 31 (Figure 4a).
In contrast, no presence of these patterns was observed for
solutions of copolymer E10S10E10 or copolymer E137S18E137 in
the concentration range analyzed, even in solutions not optically
clear. The different sizes of the observed circular patterns in
polarized spectroscopy denote the polydisperse size of the vesicles.
It is necessary to note that samples for POM experiments were
unfiltered samples provided that filtered ones did not allow
accurate measurements because of instrument resolution. Thus,
sizes of vesicles from POM are larger than those derived from
electron microscopies (between 1 and 5 µm) and even more
polydisperse. The presence of vesicular structures was further
corroborated by TEM and cryo-SEM, as seen in Figure 4b,c, in
this case using filtered solutions as for DLS. Despite the inherent
problems of TEM due to solvent evaporation during sample
preparation, which affects sizes and structures of nanostructures
in solution, the coexistence of micelles with a mean size of ca.
(30) Sony, S. S.; Sastry, N. V.; George, J.; Bohidar, H. B. Langmuir 2003,
19, 4597–4603.
(31) Kunieda, H.; Nakamura, K.; Davis, H. T.; Evans, D. F. Langmuir 1991,
7, 1915–1921.
106

Self-Assembly of OCH2CH(C6H5)-OCH2CH2 Block Copolymers Langmuir, Vol. 24, No. 14, 2008 7111
Figure 4. (a) POM image of vesicles formed by copolymer E12S10. Picture was taken at 100× magnification. (b) TEM images of vesicles (denoted
with arrows) that coexist with smaller micelles. (c) Cryo-SEM picture of polymer vesicles of copolymer E12S10. The smallest vesicles are denoted
with arrows.
Figure 5. Concentration dependence of the reciprocal apparent
hydrodynamic radii for micellar solutions of copolymers E137S18E137 at
(9) 15 and (O) 50°C and copolymer E10S10E10 at 15 °C (inset).
16 nm and some small vesicles of polydisperse size (mean size
of ca. 65 nm; see arrows in Figure 4b) can be discerned. Cryo-
SEM showed also the existence of larger vesicles (with sizes
around ca. 400 nm) at more concentrated copolymer solutions,
as seen in Figure 4c, which coexist with smaller vesicles of ca.
100 nm of diameter (see arrows). In this figure, pores created
from vesicle disintegration during sample preparation can be
also observed. Micelles were not seen because the equipment
Figure 6. Debye plots for micellar solutions of (a) copolymer E137S18E137
at (9) 15 and (O) 50°C and (b) copolymer E10S10E10 at 15 °C.
resolution was not sufficient. Then, comparing the size distributions
derived from DLS with the sizes of the structures seen by
TEM and SEM, we can assign the first peak of the DLS size
107

7112 Langmuir, Vol. 24, No. 14, 2008 Juárez et al.
Table 3. Micellar and Hydration Properties of Block Copolymers E12S10, E10S10E10, and E137S18E137
polymer T/°C A2/10 -4 mol g -2 cm 3 δt Mw mic /10 5 mol g -1 rh/nm Nw rt/nm
E10S10E10 15 4.5 1.0 4.7 20.2 202 21.5
E137S18E137 15 0.2 5.8 4.8 12.9 34 9.9
50 0.27 4.8 6.3 11.8 42 10.2
distribution of copolymer E12S10 to micelles provided that this
species lies in the typical sizes of copolymer micelles (with an
apparent radius of ca. 13 nm), whereas the other two peaks are
assigned to polymer vesicles of different sizes. In this regard,
differences in vesicle sizes between SEM, DLS, and TEM may
arise from solvent evaporation in the latter technique, which
dehydrates the polymer corona and shrinks the polymer structure.
On the other hand, polymer solutions when measured by TEM
showed the presence of polymer aggregated material, which may
be composed of the largest vesicles as a consequence of the
sample evaporation process since these were not observed by
this technique (picture not shown).
Micellar Properties of Copolymers E137S18E137 and
E10S10E10. Hydrodynamic Radii. Now we will focus on the
micellar properties of copolymers E137S18E137 and E10S10E10. Plots
of the reciprocal of the intensity average of rh,app (by using the
Stokes-Einstein relation) are shown in Figure 5 for copolymers
E137S18E137 and E10S10E10. The intercepts of these plots at c )
0 gave values of rh. Note that through eq 1 the reciprocal of rh,app
is proportional to Dapp/ηT and thus compensated for changes in
solvent viscosity and temperature. The positive slopes in this
figure are consistent with these micelles acting effectively as
hard spheres. 1 This behavior is usually accommodated by
introducing a diffusion second virial coefficient (kd) in the equation
of the straight line
D app ) D(1 + k d c + ...) (5)
The coefficient kd is related to the thermodynamic second
virial coefficient A2 by 32
mic
kd ) 2A2Mw - kf - 2V (6)
where kf is the friction coefficient and V is the specific volume
of the micelles in solution. As seen from Figure 6, the positive
term in eq 7 is dominant. Relating A2 to the effective hard-sphere
volume of the micelles, Vhs (A2 ) 4NAVhs/Mw 2 (mic) ) 33 shows that
the first term depends on the ratio Vhs/Mw mic . A2 values (Table
3) are positive in agreement with the commented micellar
behavior. On the other hand, the larger insensitivity of 1/rh,app
with changes in concentration of copolymer E10S10E10 is
associated to their higher molar masses and is a common feature
observed for elongated micelles. 4,14
Aggregation Numbers. As noted in the , the Debye equation
(eq 2) is for nonideal dilute solution of particles that are small
relative to the wavelength of the light. The Debye equation taken
to the second term, A2, could not be used to analyze the SLS data
for copolymer E137S18E137 as micellar interaction causes curvature
of the Debye plot across the concentration range investigated
(Figure 6). The fitting procedure used for these curves was based
on scattering theory for hard spheres 34 whereby the interparticle
interference factor (structure factor, S) in the scattering equation
mic
K * c ⁄(I - Is ) ) 1⁄SMw was approximated by
1⁄S ) [(1 + 2φ) 2 - φ 2 (4φ - φ 2 )](1 - φ) -4
(8)
where φ is the volume fraction of equivalent uniform spheres.
Values of φ were conveniently calculated from the volume fraction
of copolymer in the system by applying a thermodynamc
(7)
expansion factor δt )Vt/Va, where Vt is the thermodynamic volume
of a micelle (i.e., one-eighth of the volume, u, excluded by
one micelle to another) and Va is the anhydrous volume of a
micelle (Va )VMw mic /NA), where V is the partial specific volume
of the copolymer solute. The fitting parameter, δt, applies as an
effective parameter for compact micelles irrespective of their
exact structure. The method is equivalent to using the virial
expansion for the structure factor of effective hard spheres taken
to its seventh term 34 but requires just two adjustable parameters
(i.e., Mw mic and δt). Values of these two quantities are shown in
Table 3. In addition, weight-average association numbers, Nw,
of the copolymer micelles were calculated from the micellar
molecular mass derived from the fitting procedure shown above
and the weight-average molecular weight of the copolymer in
the unimer state (i.e., Nw ) Mw(micelle)/Mw(molecule); Table
1). Values of the equivalent hard-sphere radius (the thermodynamic
radius, rt) calculated from the thermodynamic volume of
the micelles (i.e., from Vt ) δtVa) are also listed in Table 3. It
is also clear that the effective hard-sphere radius, like the
hydrodynamic radius, has a more direct relation to spherical
micelles than to other shapes.
As expected, Nw values for copolymer E137S18E137 slightly
increase as the temperature rises since water becomes a poorer
solvent for the poly(oxyethylene) blocks. This is consistent with
the effect of temperature in micelles of other copoly(oxyalkylene)s.
28,35 In addition, present aggregation numbers are in
agreement with previously reported ones for this copolymer at
other temperatures. 23
TEM imaging was undertaken to directly visualize the micelle
shape. Copolymer E137S18E137 formed nearly spherical micelles
in solution (Figure 7a), whereas copolymer E10S10E10 selfassembled
into spheroidal aggregates (Figure 7b). Under TEM,
the diameters of micelles were close to those obtained by DLS.
The mean length of the major axis of the spheroids was ca. 22
nm. Nevertheless, one has to bear in mind that with TEM we
image single particles whereas DLS gives an average size
estimation, which is biased toward the larger-size end of the
population distribution. In addition, it should be pointed out that
rh for elongated micelles derived from DLS is an effective value
for “equivalent spheres”, and hence rh is not expected to equal
the ellipsoid or cylinder radius.
Rheological Behavior. Solutions of copolymers E10S10E10
and E137S18E137 were analyzed to study their rheological behavior.
Solutions of E10S10E10 (0-50% (w/v)) were investigated for gel
formation using tube inversion experiments. Immobility implies
no detectable flow over a period of hours or days. To a good
approximation, immobility in the test requires the gel to have a
yield stress higher than 30 Pa. 36 Adopting the notation used by
Hvidt et al., 36–38 we refer to this immobile phase as “hard gel”.
(32) Vink, H. J. Chem. Soc., Faraday Trans. 1 1985, 81, 1725–1730.
(33) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press:
Ithaca, NY, 1953.
(34) (a) Percus, J. K.; Yevick, G. J. J. Phys. ReV. 1958, 110, 1–13. (b) Vrij,
A. J. Chem. Phys. 1978, 69, 1742–1747. (c) Carnahan, N. F.; Starling, K. E.
J. Chem. Phys. 1969, 51, 635–636.
(35) Booth, C.; Attwood, D. Macromol. Rapid Commun. 2000, 21, 501–527.
(36) Hvidt, S.; Jørgensen, E. B.; Brown, W.; Schillén, K. J. Phys. Chem. 1994,
98, 12320–12328.
(37) Kelarakis, A.; Mingvanish, W.; Daniel, C.; Li, H.; Havredaki, V.; Heatley,
F.; Booth, C.; Hamley, I. W. Phys. Chem. Chem. Phys. 2000, 2, 2755–2763.
108

Self-Assembly of OCH2CH(C6H5)-OCH2CH2 Block Copolymers Langmuir, Vol. 24, No. 14, 2008 7113
Figure 7. TEM micrograhs of micelles formed by copolymers (a) E137S18E137 and (b) E10S10E10.
Figure 8. Aqueous solutions of copolymer E137S18E137. (a) Temperature
dependence of (9) storage (G′) and (O) loss (G″) moduli for aqueous
hard gel at 50% (w/v), frequency, f ) 1 Hz, and strain amplitude, A )
0.5%. (b) Temperature dependence of storage (G′) (9), and loss (G″),
(O), moduli at 10% (w/v), f ) 1 Hz, and A ) 0.5%.
They have used yield stress (σy) and modulus (storage and loss,
G′ and G″) to define three convenient subdivisions of the range
of behaviors observed: immobile hard gel (high σy and G′, G′
> G″), mobile soft gel (low σy and G′, G′ > G″), and mobile
sol (null σy and very low G′, G′ < G″).
Temperature Dependence of Copolymer E137S18E137. The solhard
gel boundary was already determined by tube inversion and
rheology previously, 39 but additional measurements were done
to complete its phase diagram. Figure 8a shows an example of
a temperature scan of the logarithm of the elastic modulus for
a concentrated solution above the critical gel concentration (cgc)
for this copolymer (19% (w/v), derived from the expression cgc
) 10 2 Faφc/δt, where φc ) 0.68 is the volume fraction of spherical
micelles packed in a body-centered structure. 39 On the basis of
a critical value of G″ ) 1 kPa for a hard gel, the 50% (w/v)
(38) Almgrem, M.; Brown, W.; Hvidt, S. Colloid Polym. Sci. 1995, 273, 2–15.
(39) Hamley, I. W.; Castelletto, V.; Ricardo, N. P. M. S.; Pinho, M. E. N.;
Booth, C.; Attwood, D.; Yang, Z. Polym. Int. 2007, 56, 88–92.
Figure 9. Phase diagram of copolymer E137S18E137 from (0) tube inversion
and (9, b) rheology showing the sol, soft gel, and hard gel regions.
copolymer solution is a hard gel in the whole range, with a
maximum value of G′ ≈ 16 kPa, and G′ > G″ throughout the
whole experiment. In Figure 8b, a temperature scan of G′ and
G″ at a concentration below the hard-gel boundary (10% w/v)
is also shown as an example. Other polymer concentrations in
this region below the cgc display the same profile (not shown).
Using the conditions described above, we found that at
temperatures at which G′ (1 Hz) exceeds 10 Pa the solutions are
soft gels, otherwise sols. 36–38 As illustrated, within the region of
raised modulus G′ was higher than G″, which justifies the
convenient term “soft gel” for the fluid to distinguish it from sol.
A complete phase diagram of the regions of sol, soft gel, and
hard gel defined by rheometry and tube inversion for copolymer
E137S18E137 is shown in Figure 9. These sol-gel diagrams follow
the general pattern as those published previously for other EO/
SO block copolymers. 1,2 The upper limit to the soft gel region
for the copolymer is not reached within the temperature range
investigated, consistent with the stability at high temperature of
the hard gels of this copolymer.
Frequency Dependence of Copolymer E137S18E137. To get a
more detailed picture about the rheological behavior of both
hard and soft gels, frequency scans of solution of E137S18E137
block copolymer were performed. Figure 10a shows the frequency
scan obtained for the 15% (w/v) solution of copolymer E137S18E137
at 25 °C. This type of soft gel at temperatures and concentrations
relatively near the hard-gel boundary can be assigned as defective
versions of the cubic packed hard gels (i.e., small structured
domains in an overall fluid matrix). These soft gels are
109

7114 Langmuir, Vol. 24, No. 14, 2008 Juárez et al.
Figure 10. Frequency dependence of (b) storage and (O) loss moduli
(log-log plot) for (a) 15% (w/v) aqueous solution of copolymer
E137S18E137 in the soft gel region and (b) 6% (w/v) aqueous solution of
copolymer E137S18E137 in the soft gel region at T ) 60 °C.
characterized by the almost constant value of log G′, the minimum
in log G″, and both moduli do not show a crossover point in the
measured frequency range, all of which are characteristic of hard
gels formed by cubic packing of spherical micelles, 40 although
the modulus values are low compared to those of the hard gel
(G′ ≈ 70 Pa and G″ ≈ 5Paatf ) 1 Hz, A ) 0.5%). Soft gels
of this type have been also found for other EmSnEm,EmSn, 28 and
EmGnEm 4 (where G ) phenylglycidyl unit) copolymers studied
by rheometry, and some EmBn copolymers using SAXS 41 and
Pluronics using SANS. 42
On the other hand, the scan obtained for a 6% (w/v) solution
at 60 °C differs from that of Figure 10a (Figure 10b), having very
low values of G′ (e.g., G′ ≈ 8Paatf ) 1 Hz, A ) 0.5%) and
with a moduli crossover at low frequency, corresponding to a
Maxwell fluid, at most, showing localized cubic order. This must
be a consequence of the weak attraction of spherical micelles in
water at temperatures where it is a poor solvent for the micelles.
The transition from sol to soft gel may well occur when aggregates
of spherical micelles reach the percolation threshold, yielding
sufficient structure to cause the characteristic rheological effect.
Soft gels of this type have been also identified in aqueous micellar
solutions of a wide range of copolymers including other EO/SO
copolymers, 1,2,28,40 as well as in EmPnEm (P ) propylene oxide
unit) by Hvidt and co-workers who ascribed the effect in certain
systems to the intervention of the sphere-to-rod micellar
transition, 36,38 and by Mallamace and co-workers who identified
mechanisms of percolation and packing (structural-arrest),
depending on temperature and concentration. 43,44
(40) Li, H.; Yu, G.-E.; Price, C.; Booth, C.; Fairclough, J. P. A.; Ryan, A. J.;
Mortensen, K. Langmuir 2003, 19, 1075–1081.
(41) Castelletto, V.; Caillet, C.; Fundin, J.; Hamley, I. W.; Yang, Z.; Kelarakis,
A. J. Chem. Phys. 2002, 116, 10947–10958.
(42) Prud’homme, R. K.; Wu, G.; Schneider, D. K. Langmuir 1996, 12, 4651–
4659.
(43) Lobry, L.; Micali, N.; Mallamace, M.; Liao, C.; Chen, S. H. Phys. ReV.
E 1999, 60, 7076–7087.
(44) Chen, S. H.; Chen, W. R.; Mallamace, F. Science 2003, 300, 619–622.
(45) Antonietti, M.; Forster, S. AdV. Mater. 2003, 15, 1323–1333.
Figure 11. (a) Temperature dependence of (b) storage and (O) loss
moduli (log-log plot) for a 20% (w/v) aqueous solution of copolymer
E10S10E10. (b) Frequency dependence of storage (close symbols) and
loss moduli (open symbols) (log-log plot) for (b) 20, (9) 30, and (2)
40% (w/v) aqueous solution of copolymer E10S10E10 at 15 °C.
Concentration and Temperature Dependence of Copolymer
E10S10E10. For copolymer E10S10E10, gelation was not found in
the concentration range analyzed. Concentrations in the range
5-30% (w/v) and temperatures in the range 10-45 °C were
investigated despite the turbid solution, a signal that the phaseseparation
boundary was close, as will be commented below.
For a given concentration of copolymer, the value of G′ decreased
with increasing temperature (Figure 11a), which is associated
with the water becoming a worse solvent for poly(oxyethylene),
resulting in a compression of the E-block corona and, thereby,
a decrease in the effective volume fraction of micelles in the
solution. The break in the curve of Figure 11a observed around
35 °C is related to solution-phase separation.
Frequency Dependence of Copolymer E10S10E10. The frequency
dependence of the modulus was determined for solutions of
copolymer E10S10E10 in the concentration range 5-40% (w/v)
at 25 °C. Solutions were mobile fluids over the frequency range
of our measurements. Typically, values of G″ exceeded those of
G′ over the accessible frequency range except at low frequencies
(Figure 11b). The viscoelasticity of the solution was modeled by
parallel Maxwell elements:
G ′ ) (G ∞ τ 2 ω 2 )/(1 + τ 2 ω 2 ) (9a)
G ″ ) (G ″ τω)/(1 + τ 2 ω 2 ) (9b)
where G∞ is the plateau value of G′ at high frequency, τ is
the relaxation time, and ω ) 2πf (f in hertz). A good fit required
the sum of several elements, but as illustrated in Figure 11b, the
essential features of the rheology of the system could be
reproduced using three elements, with characteristic relaxation
times in the order of 0.02-0.03, 0.2-0.4, and 10-15 ms,
respectively, for all the copolymer concentrations analyzed. The
smallest time represents a fast process characteristic of a mobile
fluid, whereas the slowest is assigned to an effect of intermicellar
interaction between elongated micelles. The slight upturn in the
scan at low frequency provided evidence of an emerging slow
110

Self-Assembly of OCH2CH(C6H5)-OCH2CH2 Block Copolymers Langmuir, Vol. 24, No. 14, 2008 7115
process, which we assign to the first stages of formation of an
elastic network, and which gives rise to the formation of the soft
gel. 13
Discussion
As commented previously, it is assumed that the parameters
that control the morphologies of the aggregates are mainly core
chain stretching, interfacial tension, and corona repulsion.
Consequently, factors that influence the above balance can be
used to control the aggregate architecture: polymer molecular
weight, relative block lengths, hydrophobic block structure,
solution conditions, and so forth. Compared to the separated
aggregation behavior of small and large colloidal spheres, micelles
and vesicles may form in dilute solution of block copolymers. 45,46
In the classical and most simple description, the factor determining
the shape of self-assembled amphiphilic structures is the size of
the hydrophobic moiety to the hydrophilic part. In particular, the
micellar structure formation can be described in terms of a critical
packing factor, φ, defined as
φ ) V
(10)
al
where V is the hydrophobic volume in the micelle core, a is the
cross-sectional area of the hydrophilic part, and l is the chain
length. 47 This model hypothesizes that changes in the amphiphile
geometry, reflected in changes in V, a, orl, affect the type of
aggregate the amphiphile may form. At a φ value of 0.5, the
model predicts formation of spherical lamellae. As the packing
factor decreases, the extent of curvature increases, and the structure
shape moves toward spherical micelle. Thus, in this work we
prepare three different styrene oxide block copolymers with
different relative block lengths and hydrophobe molecular
volumes made by styrene oxide units. Since previously studied
E11B11 and E14B10 were shown to form vesicles, 18 and provided
that styrene oxide chain possess a larger volume than propylene
oxide and butylene oxide units, we thought that copolymer E12S10
should possess a hydrophilic/hydrophobic block ratio and a
sufficient hydrophobe volume (because of the presence of an
aromatic ring in its structure) to form vesicles, as has been
corroborated. In addition, the small size of the hydrophilic block
should also favor vesicle formation provided that this is favored
when the hydrophilic surface area (in our case the hydrophilic
corona) decreases, as has been previously observed for structurally
related poly(ethylene oxide)-poly(propylene oxide) and poly-
(ethylene oxide)-poly(butylene oxide) block copolymers. 17,18 On
the other hand, structurally related block copolymer E11B8 (B )
butylene oxide unit) 11 has been pointed out to undergo micelle
elongation above 35 °C because of a severe increase in Nw and
geometrical considerations. The same behavior has been claimed
for copolymer E17S10, for which 20 °C was enough to greatly
increase Nw. 19 Nevertheless, image or other suitable techniques
were not used to detect the presence of other possible structures
in both cases, mainly vesicles in contrast to copolymers E11B11
and E14B10 for which spontaneous vesicle formation has been
demonstrated. 18
On the other hand, the previous arguments would also explain
why we did not find vesicular formation for the triblock E10S10E10:
(46) Yu, M.; Wang, H.; Zhou, X.; Yuan, P.; Yu, C. J. Am. Chem. Soc. 2007,
129, 14576–14577.
(47) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press:
New York; 1992; pp 380–384.
(48) Kelarakis, A.; Mai, S.-M.; Havredaki, V.; Nace, V. M.; Booth, C. Phys.
Chem. Chem. Phys. 2001, 3, 4037–4043.
(49) Castro, E.; Taboada, P.; Mosquera, V. J. Phys. Chem. B 2006, 110, 13113–
13123.
this copolymer would behave as a diblock E10S5 because of the
two core junctions in its structure, and not enough core volume
and hydrophobic/hydrophilic ratio would be achieved to allow
vesicle formation. Nevertheless, for this copolymer we found
that its aggregates possess a spheroidal form, as seen by TEM,
whereas for copolymer E137S18E137 micellar spheres were
observed. In fact, taking the length of an S unit to be 0.36 nm
per chain unit, 33 the average length of the fully stretched S10
block is approximately 3.6 nm. As the central S block is looped
in the micelle core for the triblock copolymer E10S10E10, the
effective length will be 1.8 nm. Consequently, this would be the
maximum possible radius for an anhydrous core of a spherical
micelle. This means that the maximum core volume would be
25 nm 3 and, given a liquid density at 15 °Cof1.15gcm -3 , then
the maximum aggregation number compatible with a spherical
compact geometry would be ca. 15. Despite considering that (a)
the S blocks possess Poisson distributions, as expected for an
ideal polymerization of an alkylene oxide, 33 (b) the copolymer
micelles possess water in the core associated with the hydroxyl
end groups of the S blocks, as demonstrated recently, 48,49 and
(c) the larger entropy penalty due to chain stretching, all of which
should alleviate further the stretching restriction, the derived Nw
value from SLS for these copolymer (Nw ) 202) is incompatible
with spherical micelles, which should be elongated, either
spheroidal or wormlike. This is not the case for copolymer
E137S18E137, for which a maximum aggregation number of 71 is
expected to be compatible with a spherical shape. In addition,
the longer E blocks increase the hydrophilic surface area
promoting spherical aggregates, as commented above. The
difference in micelle geometry between copolymer E137S18E137
and E10S10E10 also provides an explanation for the different
concentration dependences of the hydrodynamic radii and Debye
functions illustrated in Figures 5 and 6, respectively. In this
regard, the Debye function found for copolymer E137S18E137 is
typical of spherical copolymer micelles, with the curvature being
a result of interference from micelles reducing the scattering
intensity as concentration is increased. However, mass action
ensures that elongated micelles of copolymer E10S10E10 will
increase in length as concentration is increased at a given
temperature, an effect that progressively increases the scattering
intensity and thus opposes the effect of micelle interaction. On
the other hand, association numbers found for structurally related
block copolymers with butylene oxide and styrene oxide and
similar hydrophobic block length shows that, for example,
copolymers E13B10E13 50 (commercially available from Dow
Chemical Co.) and E16B10E16 51 do not form elongated micelles
in the temperature range analyzed (up to 45 °C) since their
aggregation numbers are sensibly lower than that obtained for
copolymer E10S10E10. Assuming a ratio of association numbers
between poly(oxyethylene)-poly(oxystyrene) and poly(oxyethyelene)-poly(oxybutylene)
copolymers of 3 (Nw,S/Nw,B ≈
3:1), 14 the association number 51 for E16B10E16 would involve
one of 153 for E10S10E10, values well outside the range possible
for spherical micelles, and smaller than the value found
experimentally in the present work at 15 °C. In contrast, copolymer
E135B20E135 showed the formation of spherical micelles with an
aggregation number of 17 at 25 °C, 22 as in the case of the closely
related copolymer E137S18E137 studied in the present work.
Finally, it has been shown 52 that a unifying rule for formation
of polymersomes in water involves the necessity of a hydrophilicto-total
mass ratio 25% < fhydrophilic < 35%. Cylindrical shapes
(50) Yu, G.-E.; Yang, Y.-W.; Yang, Z.; Attwood, D.; Booth, C. Langmuir
1996, 12, 3404–3412.
(51) Yu, G.-E.; Li, H.; Price, C.; Booth, C. Langmuir 2002, 18, 7756–7758.
(52) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973.
111

7116 Langmuir, Vol. 24, No. 14, 2008 Juárez et al.
can be expected for 35% < fhydrophilic < 50%, spherical micelles
for fhydrophilic < 45%, and inverted microstructures for fhydrophilic
< 25%. Although sensitivities of these rules to chain chemistry
and molecular weight have not been fully probed, the present
analyzed copolymers seem to be in agreement with these.
Conclusions
We have analyzed the micellization, gelation, and structure
of the aggregates of three poly(ethylene oxide)-polystyrene oxide
block copolymers. Two of them have similar block length but
different structures (E12S10,E10S10E10), and the other has longer
blocks (E137S18E137). In this way, we can compare the effect of
temperature and block length on the self-assembly properties.
The values of the critical micelle concentration show that the
short triblock copolymer has a cmc twice as large as that of the
related diblock. The effect is originated by entropy of the triblock
chains constrained by two block junctions in the core interface
of the aggregate, compared with only one constraint for the diblock
chain. Intensity fraction size distributions showed the existence
of several species in solutions of copolymer E12S10 whereas only
one for copolymers E10S10E10 and E137S18E137. Light scattering,
polarized light microscopy, and transmission and cryo-scanning
electron microscopies provided sufficient evidence to ensure the
coexistence of micelles and polydisperse vesicles for the most
concentrated samples of this copolymer (but even in the dilute
regime). The size of these vesicular structures varies between 60
and 500 nm as shown by TEM, DLS, and cryo-SEM. The classical
birefringent pattern of vesicles was observed by polarized light
microscopy. On the other hand, the single size distribution
observed for copolymers E10S10E10 and E137S18E137 was assigned
to elongated and spherical micelles, respectively. This assignment
was made in light of the high aggregation number derived for
the former copolymer which is incompatible with a spherical
geometry of the aggregated, as also corroborated by TEM. Tube
inversion was used to define the mobile-immobile (soft-hard
gel) phase boundaries. The phase diagram of copolymer
E137S18E137 was further completed to determine the sol-soft
gel-hard gel boundaries. In contrast, copolymers E12S10 and
E10S10E10 did not gel in the concentration range analyzed. Only
certain concentrations of copolymer E10S10E10 were analyzed by
rheometry, for which an upturn in the low-frequency range of
the stress moduli was observed, denoting an evidence of an
emerging slow process, which we assign to the first stages of
formation of an elastic network.
Acknowledgment. We thank the Ministerio de Educación y
Ciencia through projects MAT2007-61604 and Xunta de Galicia
for financial support. J.J. thanks USC for his PhD fellowship,
and E.C. thanks Xunta de Galicia for his “Anxeles Alvariño”
research contract. We are really grateful to Profs. D. Attwood
and C. Booth for providing several of the copolymer samples.
LA8004568
112

Surface Properties of Monolayers of Amphiphilic Poly(ethylene oxide)-Poly(styrene oxide)
Block Copolymers
Introduction
Josué Juárez, † Sonia Goy-López, † Adriana Cambón, † Miguel A. Valdez, ‡ Pablo Taboada,* ,† and
Víctor Mosquera †
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada, Facultad de
Física, UniVersidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain, and Departamento
de InVestigación en Polímeros y Materiales y Departamento de Física, UniVersidad de Sonora, Resales y
TransVersal, 83000 Hermosillo Sonora, Mexico
ReceiVed: May 31, 2010; ReVised Manuscript ReceiVed: July 30, 2010
The surface properties of poly(styrene oxide)-poly(ethylene oxide) block copolymers EO12SO10,EO10SO10EO10,
and EO137SO18EO137 at the air-water (a/w) and chloroform-water (c/w) interfaces have been analyzed by
Tracker drop tensiometry, Langmuir film balance, and atomic force microscopy (AFM). The kinetic adsorptions
for the block copolymer solutions at both interfaces were determined by the pendant drop technique. At the
a/w, the polymer adsorption is reduced as the polymer hydrophobicity increases. Measurements of the interfacial
rheological behavior of the copolymers showed that the adsorption layers at the a/w interface manifest obvious
solid-like properties in the whole accessible frequency range, whereas a viscous fluid-like behavior is displayed
at the c/w interface. The differences observed in the monolayer isotherms obtained for the three copolymers
arose from their different hydrophobic/hydrophilic block ratios and block lengths. In this regard, copolymer
EO137SO18EO137 displays an adsorption isotherm with the four classical regions (pancake, mushroom, brush,
and condensed states), whereas for EO12SO10 and EO10SO10EO10 copolymers, only two regions were observed.
The topographic images of copolymer films were obtained by AFM in noncontact mode. Surface circular
micelles are observed at the two surface transfer pressures studied. A micelle size decrease and an increase
in monolayer thickness are observed with an increase in transfer pressure. At the largest transfer pressure
used, elongated aggregates were observed. Aggregation numbers derived from AFM pictures were in fair
agreement with those obtained for micelles in solution, and they became larger as the SO weight fraction
increases at a certain deposition pressure. This can be a consequence of stronger attractive interactions between
the SO blocks to avoid contact with the solvent.
Amphiphilic block copolymers are an important class of
materials that have attracted considerable attention because of
their outstanding solution properties, such as their self-assembly
in the presence of a selective solvent or surface. 1 In particular,
the interfacial properties of block copolymers have received
great interest in the last years due to their role in the industrial
and biological fields. 2-4 The ability of block copolymers to
adsorb at an interface, modulate their properties, and even to
self-assemble into well-defined nanoscale structures in two
dimensions has led to their use in colloidal stability and coating
processes, 5 as steric barriers at solid surfaces in medical devices
to avoid undesired protein adhesion, 6 as stabilizers of liquid
interfaces of foams 7 and emulsions 8 to prevent coalescence and
flocculation, as templates for the rational design of large-scale
nanometer architectures in thin film configurations, 9,10 and as
precursors for nanoporous materials. 11
The self-assembly behavior and physicochemical properties
of diblock and triblock copolymers of polyoxyalkylenes in
solution have been extensively studied. 12-21 Variation of
hydrophobic monomer, block length, and architecture allows
close control of their physicochemical properties. The most
* To whom correspondence should be addressed. E-mail: pablo.taboada@
usc.es. Tel: 0034981563100, ext 14042. Fax: 0034981520676.
† Universidad de Santiago de Compostela.
‡ Universidad de Sonora.
J. Phys. Chem. C 2010, 114, 15703–15712 15703
10.1021/jp1049777 © 2010 American Chemical Society
Published on Web 08/26/2010
familiar copolymers of this type are those whose hydrophobic
block is formed by units of oxypropylene (-OCH2CH(CH3),
denoted as PO), 1,2-oxybutylene (-OCH2CH(C2H5), denoted
as BO), and oxyphenylethylene (-OCH2CH-(C6H5)), prepared
from styrene oxide (denoted as SO). These copolymers are
already commercially available from BASF, 22 Dow Chemical
Co., 23 and Goldschimdt AG, 24 respectively. Nevertheless, the
two-dimensional properties and aggregation of these classes of
copolymers at the air/aqueous interface has been only extensively
reported for poly(ethylene oxide)-poly(propylene oxide)poly(ethylene
oxide) (EO-PO-EO) block copolymers or Pluronics,
the X-star shaped EO-PO-EO copolymers known as
Tetronic copolymers (from BASF), 27,28 and poly(butylene
oxide)-poly(ethylene oxide) block copolymers (BO-EO). 29 Very
little or no report has been put into fundamentally understanding
the 2-D dimensional behavior of poly(ethyelene oxide)poly(styrene
oxide) block copolymers at interfaces. Therefore,
fundamental studies of SO-based block copolymers performed
at either the air/water or the air/organic solvent interface can
provide valuable information on their interfacial phase behavior
to further guide their possible use in different biomedical and
coating applications. To fill this gap, in the present work, we
studied the surface behavior and surface properties of three
poly(ethylene oxide)-poly(styrene oxide) block copolymers,
EO12SO10, EO10SO10EO10, and EO137SO18EO137 (where the
subscripts denote the respective block length) by dynamic
113

15704 J. Phys. Chem. C, Vol. 114, No. 37, 2010 Juárez et al.
TABLE 1: Molecular Characteristics of the Copolymers
Mn a /g mol -1
(NMR)
wt%SaMw/Mn (NMR)
a Mw/g
(GPC) mol-1 cmcb /g
dm-3 Γb max/10-6 mol m-2 EO12SO10 1660 73 1.05 1760 0.006 0.97
EO10SO10EO10 1980 55 1.06 2130 0.012 0.91
EO137SO18EO137 14 200 15 1.06 15 100 0.017 0.40
a Estimated uncertainty: Mn to (3%;wt%Sto(1%, Mw/Mn to
(0.01. Mw calculated from Mn and Mw/Mn. b Data from ref 32 at 15
°C.
interfacial tension, dilatational rheology, Langmuir monolayers,
and atomic force microscopy (AFM). The self-assembling
properties and solubilization capacity of these copolymers in
bulk aqueous solution were analyzed in recent studies, 30-32 in
which the existence of vesicular structures (polymersomes),
elongated micelles, and spherical micelles in bulk solution was
reported for copolymers EO12SO10, EO10SO10EO10, and
EO137SO18EO137, respectively. These block copolymers display
different volume-to-length ratios of the hydrophobic block,
relative lengths of the polyoxyethylene blocks, and polymer
architectures, which enable analyzing the influence of these
parameters on the block copolymer surface behavior. In this
regard, due to its lengthy EO blocks and higher hydrophilicity
(as denoted by its larger cmc values; see Table 1), copolymer
EO137SO18EO137 displays a clear and complete adsorption
isotherm in which the different conformational states of the
polymer chains (“pancake”, “mushroom”, and “brush” conformations)
are observed, with a significant viscous fluid behavior
and a spherical morphology of its aggregates at the surface film.
In contrast, the surface pressures of EO12SO10 and EO10SO10EO10
monolayers were measurable at a significantly smaller area per
molecule, and only two different clear regions can be distinguished
in their isotherms as a consequence of the reduction of
the fractional interfacial area occupied by EO segments. In
addition, copolymer EO12SO10 displays a clear rheological solidlike
behavior and its organization in elongated structures similar
to a string of beads upon self-assembly at the air-water (a/w)
interface. Finally, a comparison of the surface behavior of these
copolymers with that observed for structurally related polystyrenepoly(ethylene
oxide) (PS-PEO) block copolymers is also
established. 33,34
Experimental Section
Materials. Copolymers EO12SO10, EO10SO10EO10, and
EO137SO18EO137 were prepared by sequential oxyanionic polymerization
of styrene oxide, followed by ethylene oxide,
starting from a monofunctional or difunctional initiator to form
the hydrophobic block as described in detail by Crothers et al. 35
Table 1 shows the molecular characteristics, critical micellar
concentrations (cmc), and surface excess concentrations (Γmax)
of the copolymers. Water was double-distilled and degassed
before used.
Dynamic Interfacial Tension and Dilatational Rheology
Measurements. Pendant bubble tensiometry was used to
determine the dynamic interfacial tension and the interfacial
dilatational rheology at two different interfaces: air-water
(a/w) and chloroform-water (c/w) interfaces. The bubble was
formed at the tip of a U-shaped stainless steel needle (0.5 mm
inner diameter) immersed in an aqueous block copolymer
solution of the desired concentration, with the air bubble upward
for the (a/w) interface, whereas downward for the (c/w)
interface. Measurements were carried out in a Track tensiometer
equipment (I.T. Concept, France) adapted to determine surface
tension values in real time with an accuracy of (0.1 mN/m.
Interfacial tension and interfacial rheology estimations are based
on the digital profile of a drop image and the resolution of the
Gauss-Laplace equation. Win Drop software (I.T. Concept,
France) was used to obtain the surface tension values by means
of the axisymetric drop shape analysis.
The aqueous solutions concentrations of the EO12SO10,
EO10SO10EO10, and EO137SO18EO137 block copolymers were
prepared at a constant concentration of 2 × 10 -3 mg/mL, below
their respective cmc values. At the (a/w) interface, the analysis
of the interfacial tension (γ) measurements was followed in a
relative short time (2000 s) after the bubble was formed, and
the dilatational elastic storage (E′) and loss (E′′) moduli were
determined after 2000 s using a frequency sweep of 3.14, 2.09,
1.04, 0.52, 0.31, 0.15, 0.078, and 0.039 rad/s. All measurements
were carried out in a time interval of 400 s using a 10%
amplitude oscillation of the maximum volume drop. On the other
hand, experimental data of interfacial tension, γ, at the
chloroform-water interface were followed for 180 s, and after
this time, volume oscillations to the stabilized pendant drop
volume were applied. Storage, E′, and loss, E”, moduli were
determined in this way.
Briefly, the analysis of the viscoelastic properties of the
absorbed layer was performed by means of a sinusoidal
deformation of the drop area, A, at a defined frequency, ω,
produced by the Tracker tensiometer at a time t. Calculus of
the complex dilatational elasticity modulus E(ω) is determined
by
E(ω) )-A dy
dA
This can also be defined as a complex function, which can be
written as
where E′ and E′′ correspond to the storage and the loss moduli,
respectively, by analogy with 3-D rheology. Assuming that the
mechanical properties of the adsorbed layer follow a Maxwell
model behavior, the storage and loss moduli can be written as
follows
where E0i and ω0i (ω0i ) 2π/τ0i) are the corresponding weights
of the ith relaxation element to the total elasticity modulus E0
and the characteristic relaxation frequency, respectively; τ0i is
the characteristic relaxation time of the Maxwell model.
Adsorption Isotherms. Surface-pressure isotherms were
recorded for monolayers spread from chloroform solutions
(1 mg/mL) onto a nanopure-quality water subphase in a Langmuir-Blodgett
(LB) Teflon trough (model 611 from Nima
Technologies Ltc., Coventry, U.K.). Volumes ranging from 10
to 50 µL of either EO12SO10,EO10SO10EO10,orEO137SO18EO137
block copolymer solutions (1 mg/mL) were spread dropwise
on a Millipore water subphase with a Hamilton microsyringe.
To ensure complete evaporation of the solvent, a time lag of
(1)
E(ω) ) E'(ω) + iE''(ω) (2)
E'(ω) ) ∑ i
E''(ω) ) ∑ i
(ω/ω0i )
E0i 2
1 + (ω/ω0i ) 2
(ω/ω0i )
E0i 1 + (ω/ω0i ) 2
(3)
(4)
114

Amphiphilic PEO-PSO Block Copolymers J. Phys. Chem. C, Vol. 114, No. 37, 2010 15705
15 min was applied between the deposition of the copolymer
and the beginning of compression of the spread molecules. After
chloroform was evaporated, the surface area was compressed
by Teflon barriers at a rate of 5 mm · min -1 . The temperature
was kept constant at 25 °C with a water bath, and all
experiments were carried out inside a dust-free glass box. The
surface pressure was measured using a Platinum Wilhelmy plate
located midway between the two barriers and oriented perpendicular
to them, that is, parallel to the direction of the barrier
movement. The complete system was operated on an antivibration
table. On the other hand, Langmuir films were transferred
onto a freshly cleaved mica substrate for imaging. After
spreading the chloroform solutions, again at least 15 min was
allowed for solvent evaporation. The barriers were then
compressed to the targeted surface pressures, 5 and 11 mN/m,
respectively, at a speed of 5 mm/min. Once the targeted surface
pressure was reached and stable, the cleaved mica was lifted
up at a speed of 1 mm/min. Film transfer ratios of 0.95-1.0
were obtained. The resulting films were individually stored at
room temperature in clean closed vials.
Atomic Force Microscopy. AFM images of block copolymer
monolayers were performed on freshly cleaved mica substrates.
The measurements were performed in a JEOL instrument (model
JSPM 4210) in noncontact mode using nitride cantilevers
NSC15 from MicroMasch, U.S.A. (typical working frequency
and spring constant of 325 kHz and 40 N/m, respectively). The
AFM samples were dried in air or under a nitrogen flow when
required. Control samples (freshly cleaved mica and buffer
solution) were also investigated to exclude possible artifacts.
Topography and phase-shift data were collected in the trace and
retrace direction of the raster, respectively. The offset point was
adapted accordingly to the roughness of the sample. The scan
size was usually 500 nm (aspect ratio, 1 × 1), with a sample
line of 256 points and a step size of 1 µm. The scan rate was
tuned proportionally to the area scanned and kept within the
0.35-2 Hz range. Each sample was imaged several times at
different locations on the substrate to ensure reproducibility. In
all cases, the imaging of LB films was performed far from the
edge of the glass substrate to minimize local effects caused by
turbulent water flow at the boundary and meniscus effects during
transfer. Diameters and heights of copolymer aggregates were
determined by sectional analysis taken from the average of
several sections through the aggregates.
Results and Discussion
Some molecular characteristics of the investigated block
copolymers EO12SO10,EO10SO10EO10, and EO137SO18EO137 are
shown in Table 1. As expected, the two triblock copolymers
have higher cmc values than the diblock. This effect originates
from the entropy of the triblock chains constrained by two block
junctions in the core-shell interface of the micelle compared
to the only constrain for the diblock chain. The copolymers were
chosen to form two pairs, one with similar-sized hydrophobic
SO blocks but different polymer architectures (EO12SO10 and
EO10SO10EO10) and the other with completely different hydrophilic
EO block lengths (EO10SO10EO10 and EO137SO18EO137)
and relatively similar hydrophobic SO blocks. This was made
with the purpose of comparing the relative importance of the
polymer architecture and the hydrophilic/hydrophobic block ratio
on both polymer adsorption and surface rheological behavior
at the air/water and chloroform/water interfaces. In previous
studies, the polymer architecture, the volume-to-length ratios
of the hydrophobic block, and the relative lengths of the
polyoxyethylene blocks were found to be helpful in controlling
the micellar morphology of these copolymers. 32,36 In particular,
the coexistence of spherical micelles with vesicles (polymersomes)
was observed for copolymer EO12SO10; in contrast, the
formation of elongated and spherical micelles for copolymers
EO10SO10EO10 and EO137SO18EO137, respectively, was found
upon their self-assembly. 32
Dynamic Surface Tension
The surface tension of the copolymers as a function of time
for copolymers EO12SO10,EO10SO10EO10, and EO137SO18EO137
at a concentration of 2 × 10-3 mg/mL is illustrated in Figure
1. The low concentration used allowed us to work in relative
short time intervals to achieve a quasi-stabilization at both
air-water (a/w) and chloroform-water (c/w) interfaces, and it
also avoided complications associated with chloroform evaporation.
Nevertheless, the true equilibrium state was not attained
for any of the copolymers within 1hofadsorption because the
surface tension still continued to decrease slightly with time.
Usually, the dynamic surface tension curves can be divided
in three stages: At relative low polymer concentrations, a quasiplateau
or a very slow decrease of γ(t) can be distinguished at
short times, which corresponds to the so-called lag phase. The
postlag phase stage of the polymer adsorption process is
characterized by a rapid decrease of γ(t), which is associated
with the increased number of copolymer molecules adsorbed
at the interface and to conformational changes of the polymer
chains adsorbed at the interface. In this stage, lateral interactions
between adsorbed macromolecules stabilize the monolayer and
the adsorption of the molecules continues to be controlled by
macromolecule diffusion without appreciable desorption. Finally,
the postlag stage transforms to the final (or last) adsorption
phase, which is recognized by some slowing down of the
interfacial tension decrease rate. At this stage, the dense
adsorption layer exerts electrostatic and steric repulsion toward
newly arrived macromolecules and hinders their adsorption at
the interface. 37,38 Nevertheless, despite their hindrance, the
interfacial tension tends to continuously decrease during this
stage because of the high surface activity of the block
copolymers.
In our case, initial γ values of 66.4, 64.5, and 57.1 mN/m
were observed for copolymers EO12SO10, EO10SO10EO10, and
EO137SO18EO137, respectively (see Figure 1a), as a consequence
of the very rapid migration of copolymer molecules from the
bulk solution to the a/w interface, giving rise to a block
copolymer monolayer. For copolymer EO137SO18EO137, we
assume that the observed experimental data corresponds to the
last part of the postlag time stage and the final polymeric
adsorption stage. In contrast, for both EO12SO10 and
EO10SO10EO10, the curve profile can be divided in three stages
after an almost instantaneous surface tension decrease. We will
keep the same nomenclature (lag phase, postlag phase, and final
adsorption phase) to define the regions in these curves despite
that their true meaning corresponds to values departing from
the surface tension value of pure water. For copolymer
EO12SO10, the lag phase is longer, and the decrease of γ in the
postlag phase is faster than for copolymer EO10SO10EO10,
probably as a result of its greater hydrophobicity. Similarly, a
much larger EO block for copolymer EO137SO18EO137 if
compared to EO10SO10EO10 results in a larger surface tension
decrease in the very early stages of the adsorption process, which
we attribute to an increased repulsion between the EO chains,
giving rise to an enhancement of the surface pressure.
If the adsorption kinetics is controlled by the diffusion of
macromolecules from the bulk solution to the interface, for
115

15706 J. Phys. Chem. C, Vol. 114, No. 37, 2010 Juárez et al.
Figure 1. (a) Variation of surface tension, γ, with time at the air-water
interface for copolymers (O) EO12SO10, (b) EO10SO10EO10, and (2)
EO137SO18EO137. (b) Variation of surface concentration, Γ, with time
at the air-water interface for copolymers (s) EO12SO10, (·····)
EO10SO10EO10, and (----) EO137SO18EO137. (c) Variation of surface
tension, γ, with time at the chloroform-water interface for copolymers
(9) EO12SO10, (b) EO10SO10EO10, and (2) EO137SO18EO137.
strongly adsorbing species, this is described by the Ward-Torday
equation, 39 Γ(t) ) 2c0(Dt/π) 1/2 , where c0 is the polymer
concentration and D the bulk diffusion coefficient. A diffusioncontrolled
adsorption mechanism will be expected to give a
straight line in a plot of Γ versus t 1/2 . In these experiments, the
direct connection between Γ and t cannot be measured, but a
measured value of Π may be converted to a relative value of Γ
by using the scaling expression Π ∼ Γ y and the calculated value
of y from the adsorption isotherm experiments (see below).
Figure 1b shows such a plot for the three copolymers. The
proportionally constant was set equal to 1 (m 2 · mg -2 ) y mN · m -1 ,
which implies that the surface pressure is set to 1 mN · m -1 at
a surface concentration of 1 mg · m -2 . This value is typical for
that found for many polymers. 40,41 From this figure, it can be
observed that the expected straight line is quite steep at very
short times, but a curvature in the plot is present at larger times.
This may indicate that the diffusion-controlled adsorption
process only operates during the first few seconds of the process
and, then, is more likely controlled by a slow configurational
change and reorientation of the polymer molecules at the surface.
At this respect, a bimodal type of adsorption kinetics of Pluronic
block copolymers, including initial diffusion, followed by an
internal reorganization of the structure, was discussed recently. 42
On the other hand, there was no measurable effect on the
interfacial tension at the chloroform/water interface (c/w) due
to the adsorption of copolymer EO12SO10 (see Figure 1c). This
behavior might originate from the small amount of copolymer
at the interface due to the slow diffusion of their chains to the
interface in combination with a low bulk concentration. The
slow diffusion can be associated with the fact that chloroform
is a good solvent for both EO and SO blocks, and provided
that copolymer EO12SO10 is very hydrophobic, the copolymer
chains prefer being in the organic phase. This can be additionally
corroborated with the progressively faster decrease observed in
surface tension values at short times for the increasingly
hydrophilic copolymers EO10SO10EO10 and EO137SO18EO137.
This faster decrease also enables the development of a postlag
phase for these copolymers. In fact, for the latter copolymer,
the curve profile is very similar to that obtained at the a/w
interface, although the absolute γ values are quite different as
a result of the depression of interfacial tension due to the
presence of chloroform.
Dilatational Rheology. Analysis of the frequency dependence
of the dilational moduli allows us to get detailed information
about the type of interactions existing between polymer chains
located at the interface. Figure 2 shows the frequency dependence
of storage, E′, and loss, E′′, dilatational moduli in
semilogarithmic plots for the adsorbed layers of block copolymers
EO12SO10,EO10SO10EO10, and EO137SO18EO137 at both the
a/w and the c/w interfaces. The frequency dependence of both
E′ and E′′ is generally believed to be caused by relaxation
processes at the interface. 43,44 A satisfactory rheological description
of the adsorption layers of macromolecules may be done
by considering several characteristic relaxation frequencies (or
relaxationtimes,τ),whicharerelatedtotheadsorption-desorption
exchange of polymer chains between the surface and the
adjacent subinterface layer during dilatational perturbation and
molecular reconformation of the adsorbed layer. 38 In this way,
the frequency dependences of storage (E′) and loss (E′′) moduli
were fitted to the sum of two Maxwell elements
(ω/ω01 )
E'(ω) ) E01 2
1 + (ω/ω01 ) 2 + E (ω/ω02 )
02
2
1 + (ω/ω02 ) 2
(ω/ω01 )
E''(ω) ) E01 1 + (ω/ω01 ) 2 + E (ω/ω02 )
02
1 + (ω/ω02 ) 2
where E′ is related to lateral interactions between polymer
segments at the interface plane and is relevant for the rigidity
of interfacial film, whereas E′′ is related to molecular reorga-
(5)
(6)
116

Amphiphilic PEO-PSO Block Copolymers J. Phys. Chem. C, Vol. 114, No. 37, 2010 15707
Figure 2. Storage, E′ (closed symbols), and loss, E′′ (open symbols),
moduli as a function of frequency for copolymers (9) EO12SO10, (b)
EO10SO10EO10, and (2) EO137SO18EO137 at the (a) air-water and (b)
chloroform-water interfaces.
nization processes, such as the expulsion of polymer chains from
the interface upon compression and the interactions of polymer
molecules with adjacent liquid molecules. 45 The first characteristic
relaxation time, τ01, is usually attributed to the
adsorption-desorption exhange of molecules and polymer
segments between the surface and the adjacent subsurface layer
during dilatational perturbations of the interface; τ02 corresponds
to slower reconformations of the adsorbed macromolecules
inside the adsorption layer.
At the a/w interface, the adsorption layers manifest obvious
solid-like properties in the whole accessible frequency range
(ω ) 0.039-3.14 rad/s) with E′ >E′′, following the sequence
EO12SO10 > EO10SO10EO10 > EO137SO18EO137, and with a low
significance of dissipative processes during dilational deformations
of these block copolymer layers. These values suggest a
more fluid layer in the case of copolymer EO137SO18EO137 as a
consequence of its lower hydrophobicity (larger cmc) due to
the presence of extended oxyethyelene chains. In contrast,
copolymer EO12SO10 shows the most important solid-like
behavior, which involves the formation of tightly packed
copolymer chains at the interface to avoid contact between
hydrophobic blocks and water. Molar copolymer concentration
differences slightly affect the final dilatational moduli values
(not shown). We also point out that the storage modulus E′
slightly, but continuously, increases with frequency, whereas
TABLE 2: Characteristic Relaxation Times, τ01 and τ02, and
Characteristic Dilatational Moduli Weights of Each
Relaxation Mode, E01 and E02, of Copolymers EO12SO10,
EO10SO10EO10, and EO137SO18EO137 at the Air-Water and
Chloroform-Water Interfaces
τ01 a /s τ02 a /s E01 a /mN m -1 E02 a /mN m -1
EO12SO10 3.2 48.0
air-water
4.9 19.3
EO10SO10EO10 10.0 75.0 6.8 13.8
EO137SO18EO137 6.6 81.1 1.7
chloroform-water
8.5
EO12SO10 56.3 5.7 5.6 0.3
EO10SO10EO10 51.1 8.7 4.8 2.4
EO137SO18EO137 75.9 7.1 2.1 0.9
a Estimated uncertainty: τ01, τ01 to (1%; E01, E02 to (0.01.
the loss modulus E′′ is close to zero, in particular, for copolymer
EO137SO18EO137. This involves that the exchange processes
between the surface layer and bulk solution are relatively
important, and as the frequency becomes larger, the system does
not have enough time to reach the equilibrium and returns most
of the stored energy. Thus, the elasticity becomes larger.
Otherwise, the time is not long enough to modify the interfacial
concentration gradient through different relaxation processes at
high oscillation frequencies. As a result, the dilational moduli
increase with increasing oscillation frequencies. On the other
hand, τ01 and τ02 values at the a/w interface for copolymers
EO12SO10, EO10SO10EO10, and EO137SO18EO137 derived from
the Maxwell modelization are displayed in Table 2. In particular,
copolymer EO12SO10 possesses the shortest relaxation times τ01
and τ02, which indicates the fastest adsorption-desorption
exchange and a rapid molecular reorganization of the polymer
chains adsorbed at the interface probably as a result of its lower
molecular weight, diblock architecture, and larger hydrophobicity.
At the c/w interface, the profiles of the dilational rheology
curves display similar behavior as at the a/w interface, although
the absolute values of E01 and E02 are sensibly lower, particularly
the former ones. For this reason, it seems that a viscous fluidlike
behavior is displayed by the three copolymers at the c/w
interface. Moreover, the values obtained for the characteristic
relaxation times τ01 and intrinsic elasticity E01 decreased,
whereas τ02 and E02 increased if compared to those obtained at
the a/w interface; that is, a reverse behavior is found. This may
point out that the copolymers have been weakly adsorbed at
the c/w interface because copolymer molecules tend to stay at
the chloroform phase because chloroform is a good solvent for
both hydrophilic and hydrophobic copolymer blocks, as previously
mentioned.
Adsorption Isotherms. Monolayers of EO12SO10, EO10-
SO10EO10, and EO137SO18EO137 block copolymers were spread
on a Langmuir-Blodgett trough balance, and the Π-A isotherms
were obtained (see Figure 3a). In the present study,
copolymer EO137SO18EO137 displays a classical isotherm pattern
divided in four regions. When no pressure is exerted, copolymer
chains should lie on the interface with a flat (“pancake”)
conformation parallel to the surface plane. 46,47 The area occupied
is a function of the number of SO and EO units. Roughly, the
maximum cross-sectional area occupied by an EO unit is
13-16.5 Å 225 and that occupied by an SO unit can be taken
similar as that of a PS unit (50 Å 2 ). 48 Once hydrated, the areas
of EO and SO units increase by 8.5 Å 2 (a water molecule). Once
the compression of the EO137SO18EO137 monolayer began, the
surface pressure gradually increased until reaching an area under
compression of 7350 Å 2 /molecule. This value agrees with the
117

15708 J. Phys. Chem. C, Vol. 114, No. 37, 2010 Juárez et al.
Figure 3. (a) Surface pressure, Π, isotherms for spread monolayers
of copolymers (blue line) EO12SO10, (red line) EO10SO10EO10, and
(black line) EO137SO18EO137. The inset is an enlargement of the isotherm
corresconding to copolymers EO12SO10 and EO10SO10EO10. (b) Compressibility,
K, against surface pressure of spread monolayers of
copolymers (red line) EO12SO10, (blue line) EO10SO10EO10, and (black
line) EO137SO18EO137.
sum of the maximum transverse area occupied by all EO and
SO units of copolymer EO137SO18EO137. As the pressure
increased, the hydrophobic SO blocks, initially on the air-water
interface, were lifted away. The surface pressure at which this
phenomenon occurs is usually very low owing to the weakness
of the interaction between the aqueous medium and the
hydrophobic groups 49 (Figure 3a, region ii). The 4800 Å 2 /
molecule area corresponds to an arrangement of the molecules
that leads to an occupied area 35% lower than the original value.
From 6.1 to 9.8 mN/m, another region of a lower slope appears
due to a change in the copolymer conformation, which enables
the penetration of the hydrophilic EO chains into the subphase:
the copolymer adopts a “mushroom” conformation (Figure 3a,
region iii). In this region, there is a slight increase in the surface
pressure as the surface area is decreased, indicating that a true
first-order transition does not occur. This “pseudoplateau” is
interpreted as a rearrangement of the SO coils into “loops”
within the monolayer regime and the immersion of more EO
units in the aqueous subphase. The end of the pseudoplateau
region correponds to an area of ∼1800 Å 2 /molecule, which
indicates that the vast majority of EO units is in the subphase.
Isotherms of PS-PEO copolymers of different PEO lengths
showed that the PEO block length largely influences in the
character of this transition. 50 Further compression causes a rapid
increase in surface pressure. Block copolymer molecules gradually
become closer, the mobility of the blocks becomes restricted
because of both space limitations and the increase in lateral
interactions, and the copolymer molecules reorganize into a
“brush conformation” (Figure 3a, region iv). 51-53 In the subphase,
the EO chains entangle with neighboring copolymer
molecules, whereas at the interface, the SO blocks can form
loops and even are partially solubilized in the aqueous EO layer.
If the area is further restricted, both hydrophilic and hydrophobic
blocks become stretched (condensed state). 25 The extrapolation
of the experimental data above this pressure indicates that the
area occupied per molecule at the condensed state (AL) is
276 Å 2 .
In contrast, copolymers EO12SO10 and EO10SO10EO10 exerted
a relatively low resistance to compression at the largest and
medium areas per molecule of their isotherms (see Figure 3a),
as expected from their low molecular weight, shorter SO and
EO segments, and fair aqueous solubility at low concentrations.
The surface pressures of EO12SO10 and EO10SO10EO10 monolayers
were measurable at significantly smaller areas per
molecule than for the EO137SO18EO137 monolayer. Only two
well-defined regions can be distinguished in their isotherms.
When the area per molecule is large, the surface pressure slowly
increases as the area decreases. Above 1 and 2 mN/m for
EO12SO10 and EO10SO10EO10, respectively, a very slight pseudoplateau
might be depicted. This behavior arises from the
increasing weight fraction of SO blocks at the interface, which
reduces the fractional interfacial area occupied by EO segments.
Above 3 and 5 mN/m, respectively, a sharp rise in the surface
pressure occurs. This is consistent with a significant contribution
from the SO domains, showing no plateau or pseudoplateau at
10 mN/m. In fact, the monolayers of both block copolymers
can be considered as condensed-like films in comparison with
that obtained for EO137SO18EO137, which behaves as an expandedlike
film. 53 The area occupied per molecule at the condensed
state (AL) is 167 Å 2 for E12S10 and 135 Å 2 for EO10SO10EO10,
respectively. These values are in fair agreement with the areas
predicted from the number of SO units of EO12SO10 and
EO10SO10EO10, respectively, and support the fact that the
interface is occupied only by SO blocks. This is also confirmed
by the fact that, despite that the isotherms of the copolymers
clearly differ in their shape at low surface pressures, they merge,
becoming almost superimposable at large surface pressures.
On the other hand, the compressibility modulus (K )-1/A
× (dA/dπ)) displays local maxima for every phase transition in
the copolymers’ monolayers. As shown in Figure 3b, several
phase transitions located at ∼6.5, ∼9.5, and ∼14.0 m/Nm were
found for copolymer EO137SO18EO137. Those at ∼6.5 and ∼9.5
m/Nm are PEO-related phase transitions, which are a consequence
of the penetration of PEO chains in the subphase, leaving
the air-water interface to form a swollen three-dimensional
structure in the aqueous phase (in particular, at ∼9.5 m/mN).
After the latter peak, the subsequent minimum and additional
maximum found at larger surface pressures is a result of the
increasing repulsion between SO segments in the top layer and
PEO segments in the subphase. This facilitates the desorption
of SO blocks due to enhanced repulsions within the layer and
the formation of loops and tails, which leads to a faster
redistribution between the upper and lower regions of the layer.
On the other hand, only slight maxima at ∼2.0 m/mN and a
shoulder at ca. 6.0 m/mN are found for copolymers EO12SO10
and EO10SO10EO10. The maximum can be related to a PEO
phase transition, whereas the shoulder at 6.5 m/mN can resemble
118

Amphiphilic PEO-PSO Block Copolymers J. Phys. Chem. C, Vol. 114, No. 37, 2010 15709
Figure 4. Surface pressure isotherms plotted with respect to (a) the
average area per EO repeat unit and (b) the average area per SO repeat
unit for copolymers (blue line) EO12SO10, (red line) EO10SO10EO10,
and (black line) EO137SO18EO137.
some kind of structural rearrangement of SO blocks before
complete film condensation. The absence of the transition at
9.5 m/Nm for both EO12SO10 and EO10SO10EO10 is a consequence
of their small PEO block length.
The effect of the polymer structure can be easily visualized
by plotting the surface pressure as a function of the average
surface area per monomer. Isotherms plotted with the molecular
area normalized with respect to the number of EO and SO
segments are shown in Figure 4a,b. Figure 4a demonstrates that
all of the isotherms of the three copolymers fall on a single
curve for surface areas corresponding to the semidilute regime.
This indicates that the isotherm in this regime is mainly
dependent on the EO block length. Beyond the pseudoplateau,
all copolymers appear to be affected by the SO/EO ratio. In the
semidilute regime, the Flory coefficient of the system can be
obtained from the des Cloizeaux equation, 54 which describes
the relationship between the surface pressure and the surface
concentration (Γ) of a polymer by
Π ) CA -y ) KΓ y with y ) dν/(dν - 1) (7)
where C and K are proportionality constants, A is the molecular
area, y is the scaling exponent, d is the geometrical dimension,
and ν is the Flory coefficient used to express the radius of
gyration in terms of molecular weight (Rg ≈ M ν ) and is a
measure of the solvent quality. 55 For chains in good solvent
conditions, the exponent ν is theoretically predicted to be 0.75
for a 2D self-avoiding walk and 0.6 for a 3D self-avoiding walk.
Consequently, the theoretical exponent for the scaling of the
surface pressure with the area per molecule is y ) 3 for the 2D
semidilute regime and y ) 2.25 for the 3D semidilute regime.
Plotting the variation of the surface pressure Π versus the
inverse of the area per molecules 1/A on a double-logarithmic
scale for the three different copolymers in the intermediate
region between the dilute regime and the plateau, we have
obtained y exponents of 2.28, 2.37, and 2.54 for EO12SO10,
EO10SO10EO10, and EO137SO18EO137, respectively. This indicates
that the chain conformation is almost a 3D interpenetrated layer
for copolymers EO12SO10 and EO10SO10EO10 and a intermediate
between a purely 2D entangled layer and a 3D interpenetrated
layer for copolymer EO137SO18EO137, as also observed for
different PSO-EO block copolymers. 56 From Figure 4b, the
isotherms superimposed in the concentrated regime for the three
block copolymers, which denotes that the isotherm shape is now
determined by the SO chain length and SO/EO molar ratio.
Summarizing, if the SO block is short compared with the
EO block, as in the case of copolymer EO137SO18EO137, its role
is limited to anchoring the EO chains at the air-water interface
and it has no influence on the surface pressure isotherm or on
the segment profile of the EO chains normal to the interface.
But, as the SO block is repulsive to the EO block, as in the
case of EO12SO10 and EO10SO10EO10, this should modify
the attractive nature of the interface for the EO and, therefore,
the expected concentration profile for the EO chains, as also
occurred for PS-EO and EO-PS-EO copolymers 34,47,49,57,58 and
Pluronics 26 and Tetronics (four star-shaped ethylene oxidepropylene
oxide block copolymers). 28 The balance between these
opposite interactions will depend on the copolymer surface
density, the SO block length, and the temperature.
AFM. Besides the isotherm data, detailed insight into the
organization and morphology of the Langmuir-Blodgett monolayers
should rely on the images of their corresponding films
by AFM. We compare the morphologies of the three samples
deposited at different pressures. Figure 5 shows AFM images
of the block copolymer films on freshly cleaved mica obtained
at 5 and 11 mN/m surface pressures. Aggregation was observed
for all the copolymers investigated. The surface micelle assembly
can take place (i) during the solvent evaporation step
or (ii) during the LB tranfer. Nevertheless, dynamic lightscattering
measurements (DLS, data not shown) of the three
copolymers in chloroform did not show any evidence of micelle
formation, which implies that the formation of EO-SO surface
features in LB films are the result of spontaneous copolymer
aggregation at the air-water interface rather than a transfer of
micelles formed in the spreading solution. 59
At a surface pressure of 5 mN/m, the AFM images of the
block copolymers EO12SO10, EO10SO10EO10, and EO137-
SO18EO137 show the presence of spherical aggregates with
diameters of 6.7 ( 0.4, 7.9 ( 0.2, and 9.1 ( 0.3 nm, respectively
(Figure 5a,c,e; see Figure S1 in the Supporting Information for
size distributions). The average height of the aggregates obtained
at this surface pressure is 2.03 ( 0.01, 1.25 ( 0.01, and 1.52
( 0.01 nm for EO12SO10, EO10SO10EO10, and EO137SO18EO137,
respectively, so they can be identified as dots or circular micelles
(see Figure 6 as an example and Figure S2 in the Supporting
Informationfor height distributions). The difference in dot
heights for the three copolymers is a consequence of the
119

15710 J. Phys. Chem. C, Vol. 114, No. 37, 2010 Juárez et al.
Figure 5. Typical AFM images of Langmuir-Blodgett films of the
copolymers EO12SO10 (a, b), EO137SO18EO137 (c, d), and EO10SO10EO10
(e, f) at transfer surface pressures of 5 and 11 mN/m, respectively.
Arrows denote some elongated polymer structures.
copolymer hydrophobicity and SO/EO ratio: Assuming that the
length of an SO unit is 0.18 nm per chain unit, 60 the average
length of the fully stretched SO10 and SO18 blocks would be
1.8 and 3.3 nm. As the central block is looped in the aggregate
core in the case of the triblock copolymers, the effective length
would be 0.9 and 1.7 nm, respectively. On comparison with
the experimental values, the SO blocks of copolymers EO12SO10
and EO10SO10EO10 display a more extended perpendicular
configuration on the water surface (“brush” state) than for
copolymer EO137SO18EO137, for which a “mushroom” conformation
is predominant, confirming the surface-pressure isotherm
data.
When the transfer surface pressure rises to 11 mN/m, the
height of the EO12SO10 layer largely increases up to 5.2 ( 0.01
nm, which suggests the formation of multilayers at the a/w
interface. Formation of large aggregates is also observed as a
consequence of micelle association. Part of these supraaggregates
display an elongated morphology, which seems to
be composed of a string of beads, the beads being circular
micelles in contact each other. Similar necklace-network strctures
have been observed, for example, in Langmuir-Blodgett
films of mixed PS and PS-P2VP (P2VP, poly(2-vinylpyridine)).
61 This large-scale aggregation pattern is observed,
suggesting the possibility of a dewetting process, although we
cannot neglect the possible influence of particle aggregation
upon film drying. In the case of copolymers EO10SO10EO10 and
EO137SO18EO137, the increase in film thickness increase upon
transfer at 11 mN/m is less pronounced, with mean height values
of 1.95 ( 0.01 and 3.90 ( 0.02 nm, respectively. These values
point out that the SO blocks of both copolymers are in a full
extended perpendicular configuration (brush state) at this transfer
surface pressure. The average size of the aggregates is 7.2 (
0.1, 5.6 ( 0.3, and 7.3 ( 0.1 nm for EO12SO10, EO10SO10EO10,
and EO137SO18EO137, respectively, at this transfer pressure.
Comparing these values with those measured at a surface
pressure of 5 mN/m, we can assume that a compaction of the
aggregates formed by copolymers EO10SO10EO10 and
EO137SO18EO137 has occurred in order to accommodate all
copolymer chains at the interface. Moreover, formation of
elongated micelles is also observed at 11 mN/m for the former
copolymers (see Figure 5d,f). In fact, elongated micelles in bulk
solution have been previously found for copolymer
EO10SO10EO10, whereas for EO137SO18EO137, only spherical
micelles were detected. 32
In contrast to what was found by Deveraux et al. 60 and Moffitt
et al. 33 for PS-EO block copolymers, the deposition pressure
influences the structure of the resulting copolymer surface
aggregates. Furthermore, PS-EO copolymers show additional
surface structures other than dots (elongated structures, lamellae...)
when the EO weight contents are lower than 12%; 33
however, for SO-based copolymers, elongated structures are
Figure 6. Determination of micelle size for copolymer EO137SO18EO137 at a surface pressure of 11 mN/m through sectional analysis. X represents
the aggregate diameter and Z the aggregate height.
120

Amphiphilic PEO-PSO Block Copolymers J. Phys. Chem. C, Vol. 114, No. 37, 2010 15711
TABLE 3: Aggregate Number, Nagg, for Surface Micelles of
Copolymers EO12SO10, EO10SO10EO10, and EO137SO18EO137
Nagg
5 mN/m 11 mN/m
EO12SO10 40 144
EO10SO10EO10 29 68
EO137SO18EO137 10 36
already observed at an EO content of 27% for copolymer
EO12SO10. The lower glass transition temperature of SO (∼40
°C) compared with that of a PS (∼100 °C), which is thought to
be associated with plasticization of water molecules associated
with the ether oxygen of the SO units, 12 might involve a larger
mobility of SO blocks in the aggregates, thus enabling a certain
restructurization of the aggregates consisting of lateral interactions
between SO segments of adjacent circular aggregates.
The aggregation number (Nagg) of the copolymer aggregates
was evaluated from the AFM images in conjuction with the
Π-A isotherms. Nagg was determined by dividing the number
of micelles per unit area by the molecular area from the Π-A
isotherms at the corresponding transfer pressure. The height
profile of the micelles was generally found to be hemispheric
with the condition used to scan the samples. From Table 3, it
can be seen that Nagg increases with the increase of the SO
weight fraction at a certain deposition pressure, which can be a
consequence of stronger attractive interactions between the SO
blocks to avoid contact with the solvent. Values of Nagg are in
fair agreement with those obtained for micelles in solution. 32
The lower aggregation number of copolymer EO137SO18EO137
may arise from the steric repulsion of EO chains at the interface
due to the longer PEO blocks if compared to the other block
copolymer. Moreover, Nagg for the corresponding samples tends
to increase with the increase of the deposition pressure because
further compression will provide enough energy to overcome
repulsion between EO segments and form larger aggregates.
Conclusions
In summary, the spontaneous adsorption of block copolymers
EO12SO10,EO10SO10EO10, and EO137SO18EO137 at the air-water
interface is observed to be slowed down when the hydrophobicity
of the molecule is increased. In contrast, at the chloroformwater
interface, no measurable effect is observed for copolymer
EO12SO10 due to the slow diffusion of its chains to the interface
in combination with a low bulk concentration, the slow diffusion
associated with the fact that chloroform is a good solvent for
both EO and SO blocks. In addition, the adsorption layers at
the a/w interface manifest obvious solid-like properties in the
whole accessible frequency range with E′ >E′′, following the
sequence EO12SO10 > EO10SO10EO10 > EO137SO18EO137, and
with a low significance of dissipative processes. In contrast, a
viscous fluid-like behavior is displayed by the three copolymers
at the c/w interface. Copolymer EO137SO18EO137 displays an
adsorption isotherm with the four classical regions representing
pancake, mushroom, brush, and condensed states, with the
presence of a pseudoplateau attributed to the pancake-to-brush
transition as EO chains submerge into the aqueous subphase.
On the other hand, for EO12SO10 and EO10SO10EO10 copolymers,
only two regions are observed in their adsorption isotherms as
a consequence of their low molecular weight, short SO and EO
block lengths, and much larger SO/EO ratio. The latter involves
the disappearance of the pseudoplateau region due to the
decrease in the fractional interfacial area occupied by EO
segments. Finally, surface circular micelles are observed by
AFM pictures at two surface transfer pressures. A decrease in
micelle size and an increase in monolayer thickness are observed
with an increase in transfer pressure. In addition, at the largest
transfer pressure, elongated micelles are observed. In the case
of copolymer EO12SO10, association of micelles is also observed.
Aggregation numbers derived from AFM images increase with
the increase of the SO weight fraction at a certain deposition
pressure, which can be a consequence of stronger attractive
interactions between the SO blocks to avoid contact with the
solvent. The lower aggregation number of copolymer
EO137SO18EO137 may arise from the steric repulsion of EO
chains at the interface due to the longer PEO blocks if compared
to the other block copolymer.
Acknowledgment. The authors thank the Ministerio de
Ciencia e Innovación (MICINN) for research projects MAT
2007-6107 and Xunta de Galicia for additional financial support
under project INCITE09206020PR. S.G.-L. thanks the MICINN
for her FPI scholarship. The authors also thank Prof. D. Attwood
and C. Booth for the generous gift of the copolymers.
Supporting Information Available: Size and height distributions
of aggregates formed in Langmuir-Blodgett films
of copolymers at surface pressures of 5 and 11 mN/M. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
(1) Edens, M. W. Nonionic Surfactants, Polyoxyalkylene Block Copolymers;
Marcel Dekker: New York, 1996.
(2) Riess, G.; Cheymol, A.; Hoerner, P.; Krikorian, R. AdV. Colloid
Interface Sci. 2004, 108-109, 43–48.
(3) Tao, Y.; Ma, B.; Segalman, R. A. Macromolecules 2008, 41, 7152–
7159.
(4) Boudier, A.; Aubert-Pouëssel, A.; Gérardin, C.; Devoisselle, J. M.;
Bégu, S. Int. J. Pharm. 2009, 379, 212–217.
(5) Gilcreest, V. P.; Dawson, K. A.; Gorelov, A. V. J. Phys. Chem. B
2006, 110, 21903–21910.
(6) George, P. A.; Donose, B. C.; Cooper-White, J. J. Biomaterials
2009, 30, 2449–2456.
(7) Sedev, R.; Jachimska, B.; Khristov, K.; Malysa, K.; Exrowa, D. J.
Dispersion Sci. Technol. 1999, 20, 1759–1776.
(8) Alexandridis, P.; Lindman, B. Amphiphilic Block Copolymers: Self-
Assembly and Applications; Elsevier Science: Amsterdam, 2000.
(9) Park, S.; Wang, J. W.; Kim, B.; Russell, T. P. Nano Lett. 2008, 8,
1667–1672.
(10) Park, S.; Kim, B.; Wang, J. W.; Russell, T. P. AdV. Mater. 2008,
20, 681–685.
(11) Hillmyer, H. A. AdV. Polym. Sci. 2005, 190, 137.
(12) Crothers, M.; Attwood, D.; Collet, J. H.; Yang, Z.; Booth, C.;
Taboada, P.; Mosquera, V.; Ricardo, N. P. S.; Martini, L. G. A. Langmuir
2002, 18, 8685–8691.
(13) Yang, Z. M.; Crothers, M.; Ricardo, N. P. S.; Chaibundit, C.;
Taboada, P.; Mosquera, V.; Kelarakis, A.; Havredaki, V.; Martini, L.;
Valder, C.; Collett, J. H.; Attwood, D.; Heatley, F.; Booth, C. Langmuir
2003, 19, 943–950.
(14) Booth, C.; Attwood, D.; Price, C. Phys. Chem. Chem. Phys. 2006,
8, 3612–3622.
(15) Riess, G. Prog. Polym. Sci. 2003, 28, 1107–1170.
(16) Booth, C.; Attwood, D. Macromol. Rapid Commun. 2000, 21, 501–
527.
(17) Gangaly, R.; Awal, V. K.; Hassan, P. A. J. Colloid Interface Sci.
2007, 325, 693–700.
(18) Battaglia, G.; Ryan, A. J. J. Am. Chem. Soc. 2005, 127, 8757–
8764.
(19) Bryskhe, K.; Jansson, J.; Topgaard, D.; Schillén., K.; Olsson, U.
J. Phys. Chem. B 2004, 108, 9710–9719.
(20) Chaibundit, C.; Sumanatrakool, P.; Chinchew, S.; Kanatharana, P.;
Tattershall, C. E.; Booth, C.; Yuan, Y.-F. J. Colloid Interface Sci. 2005,
283, 544–554.
(21) Barbosa, S.; Cheema, M. A.; Taboada, P.; Mosquera, V. J. Phys.
Chem. B 2007, 111, 10920–10928.
(22) Vaughn, T. G.; suter, H. R.; Lunsted, L. G.; Kramer, M. G. J. Am.
Oil Chem. Soc. 1951, 28, 294.
(23) Nace, V. M. J. Am. Oil Chem. Soc. 1996, 73, 1–8.
121

15712 J. Phys. Chem. C, Vol. 114, No. 37, 2010 Juárez et al.
(24) Fleute-Schlachter, I. Goldschmidt Industrial Specialties, Essen,
Germany. Private communication.
(25) O′Connor, S. M.; Gehrke, S. H.; Retzinger, G. S. Langmuir 1999,
15, 2580–2585.
(26) Rippner Blomquist, B.; Wärnheim, T.; Claesson, P. M. Langmuir
2005, 21, 6373–6384.
(27) González-López, J.; Sandez-Macho, I.; Concheiro, A.; Alvarez-
Lorenzo, C. J. Phys. Chem. C 2010, 114, 1181–1189.
(28) González-López, J.; Alvarez-Lorenzo, C.; Taboada, P.; Sosnik, A.;
Sandez-Macho, I.; Concheiro, A. Langmuir 2008, 24, 10688–10697.
(29) Hodges, C. S.; Neville, F.; Konovalov, O.; Hammond, R. B.;
Gidalevitz, D.; Hamley, I. W. Langmuir 2006, 22, 8821–8825.
(30) Ricardo, N. M. P. S.; Chaibundit, C.; Yang, Z.; Attwood, D.; Booth,
C. Langmuir 2006, 22, 1301–1306.
(31) Pinho, M. E. N.; Costa, F. M. L. L.; Filho, F. B. S.; Ricardo,
N. M. P. S.; Yeates, S. G.; Attwood, D.; Booth, C. Int. J. Pharm. 2007,
328, 95–98.
(32) Juárez, J.; Taboada, P.; Valdez, M. A.; Mosquera, V. Langmuir
2008, 24, 7107–7116.
(33) Cheyne, R. B.; Moffitt, M. G. Langmuir 2005, 21, 5453–5460.
(34) Deschênes, L.; Bousmina, M.; Ritcey, A. M. Langmuir 2008, 24,
3699–3708.
(35) Crothers, M.; Zhou, Z.; Ricardo, N. P. S.; Yang, Z.; Taboada, P.;
Chaibundit, C.; Attwood, D.; Booth, C. Int. J. Pharm. 2005, 293, 91–100.
(36) Elsabahy, M.; Perron, M.-È.; Bertrand, N.; Yu, G.-e.; Leroux, J.-
C. Biomacromolecules 2007, 8, 2250–2257.
(37) Babak, V. G.; Boury, F. Colloids Surf., A 2004, 243, 33–42.
(38) Babak, V. G.; Baros, F.; Boury, F.; Desbrières, J. Colloids Surf., B
2008, 65, 43–49.
(39) Ward, A. F. H.; Tordai, L. J. Chem. Phys. 1946, 14, 453–458.
(40) Hansen, F. K.; Myrvold, R. J. Colloid Interface Sci. 1995, 176,
498.
(41) Myrvold, R.; Hansen, F. K.; Balinov, B. Colloids Surf., A 1996,
18, 453.
(42) Muñoz, M. G.; Monroy, F.; Ortega, F.; Rubio, R. G.; Langevin,
D. Langmuir 2000, 16, 1094–1101.
(43) Joos, P.; Fainerman, V. B. Dynamic Surface Phenomena; VSP:
Utrecht, The Netherlands, 1999.
(44) Kopperud, H. B. M.; Hansen, F. K. Macromolecules 2001, 34,
5635–5643.
(45) Leiva, A.; Farias, A.; Gargallo, L.; Radic, D. Eur. Polym. J. 2008,
44, 2589–2598.
(46) Haefele, T.; Kita-Tokarczyk, K.; Meier, W. Langmuir 2006, 22,
1164–1172.
(47) Kiss, E.; Keszthelyi, T.; Kormany, G.; Hakkel, O. Macromolecules
2006, 39, 9375–9384.
(48) Elbolk, T. A.; Detellier, C. J. Phys. Chem. Solids. 2006, 67, 950–
955.
(49) Hann, R. A. Molecular Structure and Monolayer Properties. In
Langmuir-Blodgett Films; Roberts, G., Ed.; Plenum Press: New York, 1990;
pp 18-23.
(50) Nivaggioli, T.; Tsao, B.; Alexandridis, P.; Hatton, A. T. Langmuir
1995, 11, 119–126.
(51) Chen, C.; Even, M. A.; Chen, Z. Macromolecules 2003, 36, 4478–
4484.
(52) Chang, L.-C.; Lin, C. Y.; Kuo, M.-W.; Gau, C.-S. J. Colloid
Interface Sci. 2005, 285, 640–652.
(53) Chang, L. C.; Chang, Y. Y.; Gau, C. S. J. Colloid Interface Sci.
2008, 322, 263–273.
(54) des Cloizeaux, J. J. Phys. (Paris) 1975, 36, 281–291.
(55) Vilanove, R.; Rondelez, F. Phys. ReV. Lett. 1980, 45, 1502.
(56) Faure, M. C.; Bassereau, P.; Lee, L. T.; Menelle, A.; Lheveder, C.
Macromolecules 1999, 32, 8538–8550.
(57) Goncalves da Silva, A. M.; Simoes Gamboa, A. L.; Matinho, J. M.
Langmuir 1998, 14, 5327–5330.
(58) Baker, S. M.; Leach, K. A.; Devereaux, C. E.; Gragson, D. E.
Macromolecules 2000, 33, 5432–5436.
(59) Cox, J. K.; Yu, K.; Constantine, B.; Eisenberg, A.; Lennox, R. B.
Langmuir 1999, 15, 7714–7718.
(60) Devereaux, C. A.; Baker, S. M. Macromolecules 2002, 35, 1921–
1927.
(61) Wen, G.; Chung, B.; Chang, T. Macromol. Rapid Commun. 2008,
29, 1248–1253.
JP1049777
122

4.3.- Appended papers.
In the present work, the solution behaviour and surface adsorption properties of three different
block copolymer (E12S10, E10S10E10 and E137S18E137), have been studied. These block copolymers
display different volume-to-length ratios of the hydrophobic block and of relative lengths of the
poly(oxyethylene) block ; and also polymer architecture may have an important role in controlling
the morphology of polymeric aggregates and surface adsorption behaviour of these copolymers.
To known the influence of such parameters, we studied the self-assembly properties and the
different structural aggregates of the three copolymers in aqueous solution.
For copolymer E12S10, coexistence of spherical micelles with vesicular structures is detected. To the
best of our knowledge, the spontaneous formation of vesicles by poly(oxystyrene)-
poly(oxyethylene) block copolymers has been first reported here. On the other hand, formation of
elongated micelles upon self-assembly of copolymer E10S10E10 in dilute solution is elucidated from
light scattering and transmission electron techniques. In the case of E137S18E137, typical spherical
micelles are observed, as expected. The micellization properties of the latter copolymer had been
already partially studied previously (89)(90), and the present study completes this gap.
On the other hand, fundamental studies of polystyrene oxide-based block copolymers performed
at either the air-water or the water-organic solvent interface can provide valuable information on
their interfacial phase behaviour to further guide their possible use in different biomedical and
coating applications. To fill this gap, in the present work, we studied the surface behaviour and
surface properties of these three copolymers, E12S10, E10S10E10 and E137S18E137, by different
techniques.
To do this work, different techniques such as surface tension, Langmuir-Blodgett through, drop
tensiometry, light scattering, transmission and scanning electron microscopies (TEM and SEM),
atomic force microscopy (AFM), polarized optical microscopy (POM) and rheometry were used.
123

4.3.1.- Relevant aspects
I. For E12S10 dilute solutions, the coexistence of spherical micelles with vesicular structures
has been observed. In addition, the spontaneous formation of vesicles by
poly(oxystyrene)-poly(oxyethylene) block copolymers is reported for the first time, as
confirmed by light scattering, polarized light microscopy, and transmission and cryo-
scanning electron microscopy data.
II. In dilute solution, the self-assembly of copolymer E10S10E10 leads to the formation of
elongated micelles as supported by light scattering and transmission electron techniques.
III. In the case of copolymer E137S18E137, typical spherical micelles are observed. Tube inversion
and rheological measurements were used to define the sol- soft- and gel-hard boundaries
of this copolymer.
IV. E12S10 and E10S10E10 copolymers did not form gels in the concentration range analysed.
However, only certain concentrations of copolymer E10S10E10 were analysed by rheometry.
From experimental data, an upturn in the low-frequency range of the stress moduli was
observed, denoting the existence of an emerging slow process, which was assigned to the
formation of an elastic network.
V. Block copolymers E12S10, E10S10E10 and E137S18E137 showed spontaneous adsorption at the
air-water interface, which slowed down when the hydrophobicity of the molecule was
increased. In contrast, at the chloroform/water interface no measurable effect is observed
for copolymer E12S10 due to the slow diffusion of its chains to the interface in combination
with a low bulk concentration; this slow diffusion is associated with the fact that
chloroform is a good solvent for both E and S blocks.
VI. Copolymer E137S18E137 displays an adsorption isotherm with the four classical regions
representing the pancake, mushroom, brush and condensed states; the presence of a
pseudo-plateau is attributed to the pancake-to-brush transition as E chains submerge into
the aqueous sub-phase. On the other hand, for E12S10 and E10S10E10 copolymers only two
regions are observed in their adsorption isotherms as a consequence of their low
molecular weights, short S and E block lengths, and much larger S/E ratio. This involves the
disappearance of the pseudo-plateau region due to the decrease in the fractional
interfacial area occupied by EO segments.
VII. According to AFM images, circular micelles are observed on Langmuir-Blodgett films of the
copolymers obtained at two surface transfer pressures. A decrease in micelle size and an
124

increase in monolayer thickness are observed with increases in transfer pressure.
Aggregation numbers derived from AFM images increase with the increase of the S weight
fraction at a certain deposition pressure, which can be a consequence of stronger
attractive interactions between the S blocks to avoid contact with the solvent.
125

Contents
5.1
5.2
5.3
5.4
5.5
5.1.1
5.1.2
5.1.3
5.1.4
5.2.1
5.2.2
5.2.3
5.2.4
5.3.1
5.3.2
5.5.1
Protein structure
Primary structure of the proteins
Secondary structure of the proteins
Tertiary structure of the proteins
Quaternary structure of the proteins
Protein aggregation
Unfolded state
Intermediate and transition state assembles
Native state
Energy landscape: protein aggregation
Amyloid fibrils
5.1.- Protein structure
Amyloid fibrils hallmarks
Kinetic fibril formation in vitro
Human serum albumin
Amyloid fibrils as biomaterials
Hen egg-white lysozyme
CHAPTER 5
Proteins
From a chemical point of view, proteins are linear heteropolymers whose backbones are
usually composed of almost 20 different monomers, as opposed to most synthetic polymers
which are composed of one or few monomers. Whilst polymers have a large extended
organization in aqueous solution, proteins fold as relatively small compacted structures. The
spatial arrangement of atoms in a protein is called conformation. The possible conformations
of a protein include any structural state which can achieve without breaking covalent bonds. A
change in conformation could occur, for example, by rotation about single bonds. The
conformations existing under a given set of conditions are usually those most
thermodynamically stable, i.e. having the lowest Gibbs energy.
127
128
129
131
132
132
133
133
134
135
135
135
138
139
141
142
127

Four structural levels are frequently considered in protein architecture. The primary structure
corresponds to the amino acid sequence. The secondary structure refers to the spatial
arrangement of amino acid residues that are nearby in the sequence (for example, α-helix and
β-sheet are elements of secondary structure). Tertiary structure refers to the spatial
arrangement of amino acid residues that are far apart in the sequence and to the pattern of
disulfide bonds. Finally, proteins containing more than one polypeptide chain exhibit a fourth
level of structural organization (each polypeptide chain in such a protein is called a subunit). In
this regard, quaternary structure refers to the spatial arrangement of subunits.
5.1.1.- Primary structure of the proteins
Proteins are linear polymers formed by the linkage of the carboxylic group of one amino acid
to the amino group of another amino acid generating a peptide bond. The formation of a
dipeptide from two amino acids is accompanied by the loss of a water molecule. A series of
amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit in a
polypeptide is called a residue. A polypeptide chain possesses polarity because its ends are
different, with an amino group at one end and a carboxylic group at the other. By convention,
the amino end is taken to be the start of the polypeptide chain, so the sequence of amino
acids in a polypeptide chain is written starting with the amino-terminal residue (4).
Figure 5.1 Polypeptide chain: structural constant backbone (inside the dotted squared box)
and lateral side lateral chains, denoted by the letter R.
As mentioned before, proteins are built by a sequence of amino acids which are chemically
connected by covalent bonds (peptide bond). A polypeptide chain is constituted of two parts: a
main chain or backbone and a complementary one constituted by side lateral chains
characteristic of each amino acid (Figure 5.1). The backbones possess a great ability to
establish hydrogen bonds: each amino acid has a carboxylic group (-C=O), that is an acceptor
of hydrogen bonds (except proline), and an amino group (-NH) which it is a donator of the
hydrogen bonds. These functional groups mutually interact to provide stability to the
biomacromolecule structure. Naturally, proteins are constituted by 20 different types of amino
128

acids whose lateral side chains change in size, form, charge, hydrophobic character and
chemical reactivity.
5.1.2.- Secondary structure of the proteins.
The secondary structure of proteins depends on four factors: i) bond length and angles of
peptidic bonds; ii) the coplanar arrangement of the substituted atoms in amide groups; iii) the
hydrogen bonding between functional –NH and C=O groups in order to maintain structural
stability; iv) the distance of the hydrogen bonds that can be formed (3). For instance, segments
of polypeptide chains are in a coiled conformation due to intramolecular interaction, i.e. the
atoms involved in amide groups must remain coplanar, and for maximal stability, each N-H
group must be hydrogen bonded to a –CO and each -CO to a –NH. Under ideal conditions,
functional groups (carboxylic and amine groups) of amino acids can form hydrogen bonds, with
an energy of 5 kcal/mol. The polypeptide chains tend to adopt up to three different
configurations that enable the formation of a maximum number of hydrogen bonds: alpha
helix, beta sheet, turns or loops (Figure 5.2).
The alpha helix (α-helix) displays a cylindrical structure (Figure 5.2a) where the polypeptidic
backbone possesses a helical conformation strongly folded into the internal cylindrical core.
The side lateral chains are extended out of the helical distribution. The α-helix is stabilised by
the hydrogen bonding along the principal backbone chain. Within this, the separation between
the amino acids is close to 1.5 Å, and each complete helical turn has 3.6 amino acids
(4)(91)(92). Alanine and leucine are strong helix favouring residues, while proline is rarely
found in helices, because in its backbone nitrogen is not to available for the hydrogen bonding
required for helix formation. The aromatic side chain of phenylalanine can sometimes
participate in weakly polar interactions (4).
The beta sheet fold (β-sheet) differs markedly from the α-helix. A polypeptide chain, called a β-
strand, in a β-sheet is almost fully extended rather than being tightly coiled as in the α-helix.
(Figure 5.2b). The distance between adjacent amino acids along the β-strands is approximately
3.5 Å, in contrast with the distance of 1.5 Å along the α-helix. A β-sheet is formed by linking
two or more β-strands by hydrogen bonds. Adjacent chains in β-sheets can run in opposite
direction (antiparallel β-sheet) or in the same direction (parallel β-sheet). In the antiparallel
arrangement, the –NH and the –CO groups of each amino acid are, respectively, hydrogen
bonded to the –CO and the –NH groups of a partner on the adjacent chain. In the parallel
arrangement, the –NH group is hydrogen bonded to the –CO group of one amino acid on the
129

adjacent strand, whereas the –CO group is hydrogen bonded to the –NH group on the amino
acid two residues farther along the chain (4)(92).
Figure 5.2 Secondary structures of the proteins a) -helix, b) β-sheet and c) turns.
In proteins, the directional changes of the polypeptide chains are led by simple structural
elements like inverse turn (Figure 5.2c), called β-turns or hairpin turns and loops. This
structure stabilized abrupt direction changes of the polypeptide chains. Contrary to α-helix and
β-sheet, turns and loops have no periodic structures (turns have a compact folded and usually
rigid structure stabilized by hydrogen bonding, while loops have an extended and disorder
structure without hydrogen bonds). These are the connecting elements that link successive
runs of the α-helix or β-sheet conformations. Particularly, β-turns connect the ends of two
130

adjacent segments of an antiparallel β-sheet. The structure is a 180 0 turn involving four amino
acid residues, with a carbonyl oxygen of the first residue forming a hydrogen bond with the
amino-group hydrogen of the fourth. The peptide groups of the central two residues do not
participate in any inter-residue hydrogen bonding (Gly and Pro residues often occur in β-turn,
the former because it is small and flexible, the later because peptide bounds involving the
amino nitrogen of proline readily assume cis configuration). Generally, β-turns are often
localized near the surface of a protein, where the peptide groups of the central two amino acid
residues in the turn can hydrogen-bonded with water. Hence, the secondary structures of
proteins make reference to highly organized regular geometries. Turns and loops invariably lie
on the surfaces of proteins and thus participate in interactions between proteins and other
molecules (91)(92).
5.1.3.- Tertiary Structure of the proteins
The overall three-dimensional arrangement of atoms in a protein is referred to as the protein
tertiary structure. Whereas the secondary structure refers to the spatial arrangement of amino
acid residues that are adjacent in a segment of a polypeptide, tertiary structure includes
longer-range aspects of the amino acid sequence. Amino acids that are far apart in the
polypeptide sequence and are in different types of secondary structure may interact within the
completely folded structure of a protein. In other words, tertiary structure emerges from the
distribution of side chains of amino acid residues. Instead, the buried parts of proteins consist
almost entirely of non-polar residues. On the other hand, charged residues usually are absent
from the inside of proteins. However, both polar and non-polar residues can be found on the
protein surface. In aqueous environment, protein folding is driven by the strong tendency of
hydrophobic residues to be excluded from water (the system is thermodynamically stable
when hydrophobic groups are clustered rather than extended into the aqueous surroundings).
The polypeptide chain therefore folds so that its hydrophobic side chains are buried and its
polar, charged chains are on the surface. Recall that many α-helix and β-strands are
amphipatic since both secondary structures have a hydrophobic face, which points into the
protein interior, and a more polar face, which points into solution. The buried domains in a
hydrophobic environment are stabilized by hydrogen bonding, pairing all the amino and
carboxylic groups. This pairing is neatly accomplished in an α-helix and β-sheet structures. Van
der Waals interactions between tightly packed hydrocarbon side chains also contribute to the
stability of the tertiary protein structure (4).
131

5.1.4.- Quaternary structure of the proteins
The quaternary structure refers to the association of several polypeptide chains (with tertiary
structure) linked by weak forces. Amongst the forces stabilising the quaternary structure we
find hydrogen bonding, hydrophobic interactions and cross-linking interactions like disulfide
bridges and metal-ion ligation. At this structural level, the protein assemblies composed of
more than one polypeptide chain are called oligomers, and each individual chain is called
subunit.
5.2.- Protein aggregation
The timescale required to protein folding in biological systems is quickly enough to avoid a
random mechanism for protein folding (93). As a general principle, the kinetic pathway of
protein folding can be described as an ordered fold reaction. In this way, the folding of only
very simple proteins has been determined at atomic level resolution to date (94), using
biophysical and theoretical experiments as a sequential and highly cooperative reaction (95).
The concept of energy landscape is used to visualize the conformational space available to
each individual polypeptide under a given condition. This theoretical formalism describes the
progressive folding of polypeptide chains along their energetic structural evolution (the energy
landscape) from an unfolded conformation to a compact native structure (Figure 5.3) (96). For
small proteins, this landscape appears to be funnel-like and represent the evolutionary
selection of a polypeptide sequence able to fold rapidly and reliably towards a unique native
state. On the other hand, larger polypeptide sequences have rougher energy landscapes, in
which there are present different populations of partially folded species that may be on- or
off-pathway to the native fold. Characterizing the multitude of populated conformational
states on this energy landscape is not only crucial for developing an understanding of the
determinants of protein folding and function, but also contains crucial clues about side-
reactions such as protein aggregation. Such characterization involves the knowledge of all
structural, kinetic and thermodynamic properties of all conformations accessible to a given
polypeptide sequence. However, it is necessary to develop analytical-high resolution tools
capable to resolve the structural fast interconversion of the species involved, as well as the
theoretical bases that permit to understand and interpret the obtained data (97).
132

5.2.1.- Unfolded state
Over the last decade, an enormous progress has been made in the description of the
conformation of the unfolded states of polypeptide chains. These important species not only
define the starting point for protein folding, but also have important roles in a variety of
biological process. When described the unfolded state of a polypeptide sequence, the first idea
in mind is a random coil structure, which lacks of specific inter-residue interactions between
the lateral chains of the amino acid sequence. However, this viewpoint has moved over recent
years as a result of the increasing number of detailed studies about the properties and
structure of denatured states under different solution conditions (98). Current views consider
that even in the presence of high denaturant concentrations, significant amounts of aliphatic
and aromatic side chains present in the native state may persist, even when the secondary
structure of the native state has been lost. On the other hand, under lesser harsh solution
conditions, also some proteins can be unfolded in the absence of denaturant agents (for
example, by mutating the sequence or changing the pH of the solution), the unfolded state has
been shown to contain significant native-like interactions with substantial native and non-
native side chain contacts which allow the existence of a residual structure. This residual
structure in the unfolded state has not only been suggested to reduce the conformational
search during protein folding, but also for some proteins may play a role in the onset of
protein aggregation of unfolded polypeptide sequences (97)(99).
5.2.2.- Intermediate and transition state ensembles
The function of the intermediate states (populated partially-folded states) during protein
folding has been a long-standing question. Supported by the observation that many small
proteins fold with apparent two-state kinetics, folding intermediates were initially thought to
be aberrant misfolds on the folding energy landscape, representing off-pathway species or
kinetics traps. Nowadays, thanks to more powerful experimental methods (Foster resonance
emission transfer fluorescence (FRET), fluorescent correlation spectroscopy or NMR
spectroscopy, amongst others) protein folding studies have revealed that partially folded
states are widely populated during the protein folding process, even in simplest proteins (97).
In this regard, a model that involves transient intermediate states in equilibrium into the
pathway of a given polypeptide chain has been suggested. Under this context, a polypeptide
chain with high molecular weight may present many intermediate states, describing a
roughness energy landscape for its particular folding pathway. For example, larger polypeptide
chains have a higher tendency to collapse under specific solution conditions, leading to the
formation of compact states, which can contain substantial native-like structure. Structural
133

eorganization as a consequence of inter-residue contact (including both native and non
native-like interactions) of these compact states may involve high-energy barriers, leading to
the transient population of partially intermediate states. Such species can be either productive
for on-pathway folding until reaching the native state; or they can be trapped in such a manner
that the native structure cannot be reached without substantial organizational events (off-
pathway).
5.2.3.- Native state
Currently, it is known that the conformational states of proteins are structural dynamic
processes, i.e. even at native conditions, structured proteins have access to a manifold of near-
native conformations, in which protein native structures show fluctuations around the minimal
energy conformation (100). Such structural movements are essential in order to encompass
their function, such as enzyme catalysis or ligand binding, as well as site-to site communication
within the globular protein fold or between proteins subunits.
Figure 5.3 Schematic illustration of the energy landscape for protein folding and aggregation.
a) The surface illustrates the roughness of the protein landscape, showing the multitude of
conformational states available to a polypeptide chain. The conformational search of a single
polypeptide chain to a functional monomer can be described in a simple folding funnel (light
grey), intermolecular association increased the roughness of the energy landscape (dark grey),
b) Proposed pathways related to the conformational states shown in a) populated on the
combined folding and aggregation energy landscape (97).
134

5.2.4.- Energy landscape: protein aggregation
The schematic diagram in Figure 5.3 attempts to depict the complexity of the protein folding
and aggregation energy landscapes as previously mentioned; at the same time, it makes an
effort to illustrate the wide variety of different conformational states with minimum energies
and the large diversity of folding pathways available for each polypeptide chain as it
circumnavigates the landscape. Regarding the in-depth knowledge of the folding landscape of
simple, single domain monomeric proteins, and the conformational states accessible to
polypeptide oligomers, relatively little is understood. Energy minima on the aggregation side of
the energy landscape might be poorly defined, as a result of broad ensembles of oligomeric
states of similar energy that are rapidly interconverting, but could also be highly defined, as
might be expected for higher order species such protofibrils or fibrils. For example, the energy
minimum of mature amyloid fibrils might be deeper and sharper that those of native
monomeric proteins, as suggested by the rigidity of the amyloid fold, as well as by the
nucleation-dependent polymerisation mechanism (101). Here, however, even under the same
solution conditions a multitude of fibril morphologies can be formed simultaneously,
highlighting the complexity and multiplicity of the aggregation pathway (102)(103).
5.3.- Amyloid fibrils
Into the context of the energy landscape, the observation that virtually any protein sequence
can form amyloid fibrils given the appropriate solution conditions led to the suggestion that
the amyloid fold is the universal global free energy minimum of all polypeptide chains that
may assemble by generic mechanisms governed by the physicochemical properties of the
polypeptide chain.
5.3.1. Amyloid fibrils hallmarks
Amyloid comes from the Greek word amylon which means starch. Amyloid was coined initially
in a botanical context by Scheiden (104). Virchow and others researches used the term
amyloid into medicine to describe human pathogenic deposits that stain with sulphuric acid
and iodine solution, similarly to starch staining. Although these deposits do not primarily
consist of polysaccharides, they were found to be a proteinaceus, nowadays the definitions of
amyloid fibrils depend on the context of their use. In pathological diagnosis, amyloid had been
defined as extracellular deposits of proteins fibrils with a characteristic appearance in electron
microscope, a typical X-ray diffraction pattern, and a strong affinity for Congo red dye (105).
135

In vivo, the self-assembly of proteins into highly ordered, β-sheet enriched, fibrillar, insoluble,
supramolecular structures known as amyloid fibrils (or amyloid-like fibrils, term referring to
protein fibrils that not associated with any disease) is linked to the onset of a growing number
of human disorders, including Alzheirmer’s, Parkinson’s, Huntington´s diseases, spongiform
encephalopathies, and type II diabetes mellitus. On the other hand, the controlled self-
assembly of proteins and peptides into amyloid-like structures may constitute an attractive
alternative to develop nanomaterials (106)(107).
Amyloid fibrils can be obtained in vitro through different ways by adjusting different external
parameters as, for example, pH, ionic strength, and temperature, and cosolute or cosolvent
additions. This means that the formation of amyloid-like conformations is not restricted to
peptide chains of specific length or having a particular primary or secondary structure; this
suggests that the conformational shift into β-sheet rich is energetically preferred. The
characterization of the fibrillation process of proteins in vitro has largely been focused on
biophysical characterization in order to determine the structure of the fibril, and on
biochemical studies to get into the mechanism and kinetics of the process. Also, through
techniques such X-ray diffraction, electron microscopy, and solid state NMR spectroscopy we
now have a good understanding of the core architecture of individual fibrils too.
In general, amyloid fibrils possess distinct physical properties as: i) they are characterized by a
cross-β sheet structure, where individual β-strands are perpendicular and each β-sheet is
parallel to the fibril axis (Figure 5.4). The X-ray diffraction pattern shows two characteristic
reflections at 4.7 Å and 10-11 Å, corresponding to the inter-strand and stacking distances
between individual β-sheets, respectively; ii) amyloids bind to histological dyes such as Congo
red (CR) and Thioflavin T (ThT). After binding to CR, an apple-green birefringence under cross-
polarized light is observed. CR and ThT dyes, however, do not bind to monomeric proteins or
peptides. These dyes fluoresce when they bind to β-sheet rich fibrils and are, therefore, useful
for spectroscopic monitoring of fibril´s growth and kinetics; iii) under electron microscope
(EM), amyloid fibrils appear to be few micrometers long, non-branched filaments, with 7-12
nm diameter. In a majority of cases, amyloids are composed of 2-4 protofilaments that are
either helically twisted or laterally associated with each other forming higher order fibrils; iv)
amyloids are resistant to heat, wide ranges of pH and proteases (107)-(113).
Over the past several years, soluble proteins and protein fragments self-assembled into
amyloid aggregates have attracted the interest of researchers from diverse fields. From a
136

molecular medicine viewpoint, protein deposition is an undesired process associated with
several human diseases. Hence, different questions must be arisen such as why self-
association of a misfolded protein results in organ dysfunction and neurodegeneration?, what
are the toxic species?, and whether aggregates of all proteins are toxic or not?. At the
molecular level, fundamental research has been directed towards obtaining a rational
understanding of the physico-chemical principles underlying protein misfolding and self-
assembly. However, given the complexity of the molecular mechanisms driving amyloid
aggregation, a frequently used strategy in the field consists of design polypeptide model
systems to allow biophysical characterization and the study of their aggregation process (114)-
(118). This strategy permits the analysis of the relationship between the amino acid sequence
of the polypeptide chain and its structural stability during the conformational transition.
Figure 5.4 Schematic representation of an amyloid fibril structure: a) β-strands perpendicular
to the fibre axis; the β-sheet structure arises from the hydrogen bonding between lateral side
chains of amino acids; b) cross-β diffraction pattern observed in amyloid fibrils. Arrows
indicate the positions of the cross-β reflection on the meridian at 4,7 Å (white) and on the
equator at 10 Å (black) (112).
a) b)
Several studies have shown that the destabilization of the polypeptide sequence per se is not
enough to bring the self-assembly process up to form amyloid fibrils (119)(120). Additionally,
the sudden conformational transition undergone by proteins in their native state to form
amyloid fibrils rich in β-sheets is favoured only if the amino acid sequence increase its ability to
form β-sheet structures. In most of these systems, the structural transitions from α-helix to β-
structures are originated by applying external heat energy. This phenomenon involves a
number of relatively weak non-covalent molecular forces: hydrophobic, electrostatic, and
137

hydrogen bonds mainly. Molecular simulations (109) demonstrated that hydrophobic
interactions play an important role in promoting protein self-assembly. Meanwhile, for small
peptides the β-sheet aggregates are stabilized by backbone hydrogen bonds, as well as by
specific side-chain interactions such as hydrophobic stacking of polar side chains and
electrostatic interaction.
5.3.2.- Kinetic fibril formation in vitro
The generic nature of the aggregation process has enabled extensive studies of the transition
between soluble precursor states and insoluble amyloid fibril in vitro (121). The aggregation
into amyloid structure of globular proteins has been shown to require a conformational
transition process before reaching the amyloid fibre structure. To carry out this process, the
native conformation of globular proteins usually must be destabilised by the addition of
denaturants agents, low pH, high temperature, truncation/mutation and so (122)-(124).
Specific protein destabilisation results in an increased population of partially folded
conformations exposing aggregation-prone regions that are usually protected in the native
state and, thus, enhancing the probability of non-native intermolecular interactions.
At present, the proposed models to describe the formation process of amyloid fibrils include
monitoring the fibrillogenesis by high-resolution microscopic and spectroscopic tools, and by
the binding of specific dyes to amyloid fibrils. The most common model for explaining the
amyloid formation is the nucleation-dependent polymerization model. In such a model, the
kinetic pathway from the native structure to amyloid fibrils occurs by a nucleation-aggregation
process. The nucleation step (also known as lag phase) is the factor that determines the rate of
amyloid formation by the slow generation of nuclei under successive thermodynamically
unfavourable steps; these nuclei aggregate when a critical protein concentration value has
been exceeded by a small amount; hence, the process is concentration-dependent. The lag
phase precedes a period of exponential growth that corresponds to fibril elongation,
characterised by a sudden fibre growth as a consequence of the addition of protein monomers
or the association of other competent species; this step is called elongation phase. In contrast,
another proposed model of fibrillogenesis is the “downhill polymerization” process, which
consists of a continuum aggregate state without requiring a nucleation step. A relatively easy
way to observe the fibrillation kinetics is by using an amyloid specific binding dye such
Thioflavin T (ThT). ThT increases its fluorescence intensity upon binding to amyloid fibrils when
using an excitation wavelength of 440 nm, and collecting the emission intensity at 480 nm.
Figure 5.5 shown the kinetic formation of amyloid fibril followed by ThT binding. Figure 5.5 a)
138

shows the typical nucleation-dependent polymerization characterised by a sudden increase of
of ThT fluorescence as a function time; once the lag phase is overcome (the lag phase is
observed as the region where no appreciable changes in ThT fluorescence intensity take
place). In contrast, with the non nucleation-dependent polymerization (downhill
polymerization, Figure 5.5b) shows a marked and continuous ThT fluorescence intensity
increase (125)-(129).
In vitro, the aggregation process of polypeptide chains into fibrillar structures can be also
monitored by means of other analytical tools such as circular dichroism (CD), infrared
spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR), light scattering,
fluorescence spectroscopy (ThT), or imaging techniques such as TEM, SEM or AFM.
Figure 5.5 ThT fluorescence intensity as a function of time following a: a) nucleation-
dependent polymerization, and b) a non-nucleation-dependent polymerization process.
5.4.- Human serum albumin (HSA)
HSA is a globular protein constituted by 585 amino acids in a single polypeptide chain with a
molecular weight of 67,000 Da. Human serum albumin is the most abundant protein in blood
plasma, it plays a special role in transporting endogenous and exogenous metabolites through
the vascular system and also maintains the pH and osmotic pressure of plasma (130). The
protein structure contains 17 disulfide bridges giving rigidity and stability to the protein
structure. HSA has one free –SH group, which leads to protein dimerization and high order
association. Accordingly to X-ray crystallography data, the secondary structure of HSA
molecule is composed of 67% -helix, no -sheet, 10% turn, and 23% extended chain
(131)(132). On the other hand, in agreement with CD spectroscopy data, the secondary
139

structure of HSA molecule at physiological solution conditions is composed of 67% -helix, 5%
-sheet, 12% turn, and 17% extended chain (133). The three-dimensional configuration of
serum albumin is divided into three major domains (labelled as I, I and III), and each domain
are composed of two subdomains that possess common structural motifs, which are
predominantly helical and cross-linked by the disulfide bridges (134)(135).
The HSA structure can be influenced by several factors as pH, temperature, cosolvents... It is
known that HSA molecule shows a number of well-defined pH-dependent structural
transitions, (Figure 5.6) (136): E form (below pH 2.7), F form (pH 2.7-4.3), N form (pH 4.3-8), B
form (pH 8-10) and A form (over pH 10) (134)-(137). On the other hand, during its thermal
unfolding process, three states can be distinguished: the native form, the reversible unfolded
form (at 65 0 C) and the irreversible unfolded form (at 74 0 C) (120). Both unfolding processes
give rise to aggregation-prone conformational changes of both secondary and tertiary
structures, allowing the formation of amorphous aggregates, intermediate states, oligomers
and amyloid fibrils.
Due to the physiological importance of human serum albumin as a carrier protein and blood
pressure regulator and its propensity to easily aggregate in vitro, HSA has become a good
model for protein aggregation studies. Moreover, as the phenomenon of protein aggregation
appears to reflect certain generic “polymeric” features of proteins (138), the study of protein
aggregation mechanisms in model systems is extremely useful to gain a better understanding
about the molecular mechanisms of disease-associated amyloidogenesis.
In this way, in this work we show the propensity of HSA to form amyloid fibrils and other types
of aggregates under different thermal and solvent conditions (changing the solution pH,
solution ionic strength and solvent composition), allowing the modulation of electrostatic,
hydrophobic and hydration interactions between protein molecules and aggregates (139)-
(141). The kinetic aggregation, protein conformational changes upon self-assembly, and
structure of the different intermediates on the fibrillation pathway were determined by means
of ThT fluorescence and CR absorbance; far and near-UV-Vis CD; tryptophan fluorescence;
Fourier transform infrared spectroscopy; X-ray diffraction; and TEM, SEM, AFM and optical
microscopy. We have observed that the fibril formation is largely affected by electrostatic
shielding: at physiological pH, fibrillation is progressively more efficient and faster in the
presence of up to 50 mM NaCl; meanwhile, at larger salt concentrations, excessive charge
shielding and further enhancement of the solution hydrophobicity might involve a change in
140

the energy landscape of the aggregation process, which makes the fibrillation process difficult.
In contrast, under acidic conditions a continuous progressive enhancement of HSA fibrillation
is observed as the electrolyte concentration in solution increases. Both the distinct protein
ionization state and the initial structural states of the protein before incubation may be the
origin of this behavior. According to ThT fluorescence data, the fibrillation kinetics of HSA does
not show a lag phase except at pH 3.0 in the absence of added salt. The HSA fibrils obtained at
the end of the fibrillation process show structural features typical of classical amyloid fibers, as
denoted by XRD, CD and TEM techniques. Finally, we describe the physical and structural
properties of additional supraself-assembled structures of HSA under solution conditions in
which amyloid fibrils are formed. We have detected the formation of ordered aggregates of
amyloid fibrils, i.e. spherulites, by means of polarized optical microscopy, laser confocal
microscopy and TEM. These structures possess a radial arrangement of the fibrils around a
disorganized protein core, and have sizes of several micrometers.
Figure 5.6 pH conditions and helical contents of the five organized forms of albumin including
crystal structure of the N form, and the proposed configuration of the F and E forms (136).
5.5.- Amyloid fibrils as biomaterials
The bottom-up strategy takes advantage of the functionality of molecular materials and
molecular recognition properties for self-assembly (142). The construction of functional
nanomaterials and components through the bottom-up strategy has potential applications in
different areas as electronics, tissue engineering, drug delivery, sensing… (143)(144). In this
regard, self-assembly properties of biomacromolecules, as phospholipids, DNA, peptides, and
proteins, can be an alternative for the obtention of new materials.
141

The physical and chemical properties of proteins are an attractive alternative for the
construction of nanoestructured materials, providing an extraordinary array of functionalities
that could be exploited in fields such as biomedicine, biotechnology, materials science, and
nanotechnology (145). Protein amyloids are excellent candidates for the fabrication of
molecular nanobiomaterials, such wires, layers, gels, scaffolds, templates, and liquid crystals,
using the bottom-up strategy as a result of their outstanding physico-chemical properties
(great stiffness, stable against heat and denaturants, resistant to proteases, amongst others)
structural compatibility, nanoscale dimensions and efficient assembly into well-defined
ultrastructures (146)(147).
On the other hand, metal nanoparticles (NP) have received considerable attention during last
decade because of their particular optical, electronic, magnetic and catalytic properties and
their important applications in many fields such as nanosensors, catalysis, biomedicine,
biological, labelling, and surface-enhanced Raman scattering (SERS). To optimize and extend
the applications of metal NPs, methods must be developed to control the assembly and
organization of these nanomaterials. NPs in ordered arrays provide optical and electronic
properties that are distinct compared to individual particles or disorganized macroscale
agglomerations. In this way, the supramolecular structure of amyloid fibrils can be used to
organize nanoparticles into predefined, topologically intricate nanostructures, or synthesize
miscellaneous materials in order to control the properties of nanoparticle assemblies for
potential applications in electronic, optical, and chemical devices. In particular, functionalizing
one-dimensional (1D) supporting biomaterials with metal NPs that combine the properties of
two functional materials, such as the high conductivity, surface area or the precise chemical
functionality of the biotemplate, and the unique plasmonic or catalytic properties of the metal
NPs widen their range of applications and, therefore, their important role in nanoscience and
nanotechnology (148)-(151).
5.5.1 Hen egg-white lysozyme
Hen egg-white lysozyme (HEWL) is a relatively small globular, monomeric protein. HEWL
consist of a polypeptide chain with 129 amino acid residues with four disulfide bonds, and it
folds into two structural domains, the α and β ones. The α domain contains a core of
hydrophobic side chains that are packed closely together (152); this domain consists of three
long helices A (5-15), B (25-35) and C (89-99) (153). In contrast, the β domain of the protein
does not display a similar hydrophobic core; instead, hydrogen bonds and a number of small
hydrophobic clusters appear to be responsible for defining its tertiary fold, and there is also an
142

exposed loop region. The secondary structure of lysozyme dissolved in water is composed of
30% -helix, 18% -sheet, 22% turn, and 30% unordered structure (153)-(155). It has been also
found that the HEWL easily forms amyloid fibrils under several denaturant solution conditions,
such acidic conditions and elevated temperature (152)(155). In this regard, it has been
proposed that fragment 57-107 is a highly amyloidogenic region, and probably the conversion
to the β-sheet structure of the C subdomain is involved in the amyloid fibril formation rather
than the other subdomains A and B (152)(153).
It is known that the proteins have multiple potential ion-binding sites within its aminoacid
sequence; therefore, protein fibrils can be used in a metallization reaction under relative harsh
conditions. In this regard, we take advantage about the spontaneous self-assembly of both
HSA and HEWL into amyloid fibrils under fibrillation solution conditions, which enables the use
of these fibrils as a template for the construction of 1-D hybrid material, in particular, gold and
Fe3O4 nanowires. The inherent structural differences between HSA and HEWL fibrils, such as
strength, length and stability make lysozyme (Lys) fibers (with widths of 4-10 nm, lengths 0.1-2
μm) most suitable for their use in the obtention of 1-D nanowires.
In this work, gold nanowires were obtained by seeded-growth process. The degree of metal
coverage of the biotemplate was controlled by sequential addition of salt gold growth
solutions. The hybrid metallic fibrils have been proved to be useful as catalytic substrates,
providing a superior catalytical activity when they are incorporated in the reduction reaction of
p-nitrophenol to p-aminophenol in the presence of NaBH4. The reaction rates obtained were
much larger than those reported for other gold hybrid materials such as Au nanoparticle
networks, PAMAM-dendrimer-suported spherical nNPs, solid 1-D Au nanobelts, and nanorods,
polymer micelles-supported Au NPs or 1-D assemblies of Au NPs (156)-(159). On the other
hand, to obtain iron oxide wires, we in situ synthesize Fe3O4 NPs by precipitation in an alkaline
medium by using the electrostatic interaction of iron ions (Fe +2 and Fe +3 ) and the negative
charged amino acid residues (mainly aspartic and glutamic acid) located on the protein fibril
surfaces. Complete magnetic coating on the protein fibril surface was obtained by sequential
nanoprecipitation in situ of Fe +2 and Fe +3 ions (1:2 molar ratio), in the presence of ammonium
hydroxide (NH4OH). Because of their large magnetic properties, the 1-dimensional magnetic
nanowires can serve as efficient magnetic resonance image (MRI) contrast agents, with
transverse relaxativities larger than those obtained previously for other 1-D nanostructures
derived by alternative methods (160)-(162).
143

Contents
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
CHAPTER 6
Papers on amyloid fibrils
Existence of different structural intermediates on the
fibrillation pathway of human serum albumin
Influence of electrostatic interactions on the fibrillation
process of human serum albumin
Additional supra-self-assembly of human serum albumin
under amyloid-like-forming solution conditions
Hydration effects on the fibrillation process of globular
protein: The case of human serum albumin
Obtention of metallic nanowires by protein biotemplating
and their catalytic application
One-dimensional magnetic nanowires obtained by protein
fibril biotemplating
Relevant aspects on the HSA fibril formation
Relevant aspects on the gold and magnetic nanowires
147
165
175
185
199
207
215
216
145

Biophysical Journal Volume 96 March 2009 2353–2370 2353
Existence of Different Structural Intermediates on the Fibrillation Pathway
of Human Serum Albumin
Josué Juárez, Pablo Taboada,* and Víctor Mosquera
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada, Facultad de Física, Universidad de Santiago de
Compostela, Santiago de Compostela, Spain
ABSTRACT The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different
solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different
intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance;
far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray
diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several
days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in
the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require
a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the b-sheet (up to
26%) and unordered conformation at the expense of a-helical conformation, as revealed by ThT fluorescence and circular
dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution
conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as
oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place
through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric
species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on
solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long
enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel
arrangement of b-strands forming the b-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation
times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures,
laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align
within the immediate vicinity of the fibers. This filament þ monomers/oligomers scenario is an alternative pathway to the otherwise
dominant filament þ filament manner of the protein fibril’s lateral growth. Conformational preferences for a certain pathway
to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.
INTRODUCTION
b-Sheet-based assemblies have attracted much interest from bonding; and 3), reach an alternative ‘‘nonnative’’ global
multidisciplinary researchers because of their association free energy minimum (10). Therefore, investigators have
with a variety of diseases and their emerging potential in emphasized understanding and inhibiting amyloid formation
material science and biotechnology (1,2). Protein misfolding more so than amyloid dissociation and clearance. Although
and self-assembly into highly ordered b-sheet-rich fibrillar the stability of b-sheet-rich amyloid fibrils against proteases,
assemblies known as amyloid fibrils are common features acids, and chemical denaturants has been shown, increasing
of a growing class of systemic and neurodegenerative evidence from human (11) and in vitro studies indicates that
diseases, including Alzheimer’s, Parkinson’s, and Hunting- a dynamic structure exists within amyloid fibrils and
ton’s diseases; senile systemic amyloidoses; type II diabetes suggests that the process of amyloid formation is reversible
(3,4); and many others. The ability to fibrillate is independent (12). These findings, along with the fact that strategies aimed
of the original native structure of the protein, whose amino at stabilizing amyloid fibrils and/or accelerating their clear-
acid sequence primarily appears to play a key role in terms ance seem to reverse the disease phenotype (13,14), suggest
of filament arrangement (5), fibrillation kinetics (6), and that a detailed understanding of the formation, stability, and
overall yield and stability of the fibrils (7,8). Fibrillation dynamic behavior of amyloid fibrils is critically important to
originates under conditions in which proteins are partially the development of therapeutic strategies for amyloid
destabilized or completely unfolded (9), and the formation diseases. Thus, the reversible untangling of amyloid archi-
of the amyloid fibrils reflects an alternative to the native tecture and intrafibrillar packing of the b-pleated sheets is
packing conformational struggle of a polypeptide chain to a key issue to consider in designing inhibitors of fibrillar
1), reduce its surface-accessible area; 2), saturate hydrogen growth (15), and insights into the fibrillar assembly mechanisms
may help elucidate the etiology of the ‘‘prion
Submitted September30, 2008, and acceptedfor publication December1, 2008.
*Correspondence: pablo.taboada@usc.es
Editor: Ruth Nussinov.
Ó 2009 by the Biophysical Society
diseases’’, provided that the subtle structural differences
underlying the puzzling phenomenon of ‘‘prion strains’’
can be understood (16).
0006-3495/09/03/2353/18 $2.00 doi: 10.1016/j.bpj.2008.12.3901
147

2354 Juárez et al.
Because of the physiological importance of human serum
albumin (HSA) as a carrier protein and blood pressure regulator,
and its propensity to easily aggregate in vitro, HSA has
become a good model for protein aggregation studies. Moreover,
as the phenomenon of protein aggregation appears to
reflect certain generic ‘‘polymeric’’ features of proteins
(17), studying mechanisms of protein aggregation in model
systems is extremely useful for gaining a better understanding
of the molecular mechanisms of disease-associated
amyloidogenesis.
Under physiological conditions, HSA consists of 585
amino acids in a single polypeptide chain, with a globular
structure composed of three main domains that are loosely
joined together through physical forces and six subdomains
that are wrapped by disulfide bonds. The protein contains
17 disulfide bridges and one free SH group, which facilitates
dimerization and also influences higher-order association.
Native HSA lacks any properties that suggest a predisposition
to form amyloid fibrils, since most of its sequence (>60%) is
arranged in an a-helix structure, with subsequent tightening
of its structure through intramolecular interactions such as
hydrogen bonds. Therefore, serum albumin aggregation is
promoted under conditions that favor partly destabilized
monomers and dimers, such as low pH, high temperature,
and the presence of chemical denaturants (18). In a recent
report (19), we showed that partially destabilized HSA molecules
form amyloid-like fibrils and other types of aggregates
under different solution conditions. These fibrils feature the
structural characteristics of amyloids: x-ray diffraction
(XRD) patterns, affinity to Congo Red (CR) and thioflavin
T (ThT), birefringence, and high stability. We now extend
that previous work to shed further light on the kinetics and
hierarchical assembly of HSA fibril formation, linking the
morphological structural transitions of aggregated protein
intermediates to conformational events on protein structure
as analyzed by means of different biophysical and spectroscopic
methods. In this way, we present a systematic investigation
of the relationship between protein conformation
and the amyloid-like self-assembly pathway for HSA under
different solution conditions. To this end, we incubated the
protein under different thermal and solvent conditions and
analyzed the protein conformation changes upon incubation.
We additionally imaged different structural intermediates on
the HSA fibrillation pathway depending on solution conditions.
In this way, we sought to uncover the structural
features underlying the formation of possibly cytotoxic
HSA assemblies.
MATERIALS AND METHODS
Materials
HSA (70024-90-7), CR, and ThT were obtained from Sigma (St. Louis,
MO) and used as received. All other chemicals were of the highest purity
available.
Biophysical Journal 96(6) 2353–2370
Preparation of HSA solutions
Protein was used after further purification by liquid chromatography using
a Superdex 75 column equilibrated with 0.01 M phosphate. Experiments
were carried out using double-distilled, deionized, and degassed water.
The buffer solutions used were glycine þ HCl (I ¼ 0.01 M) for pH 3.0,
and sodium monophosphate-sodium diphosphate for pH 7.4 (I ¼ 0.01 M),
respectively. HSA was dissolved in each buffer solution to a final concentration
of typically 20 mg/mL and dialyzed extensively against the proper
buffer. Protein concentration was determined spectrophotometrically using
a molar absorption coefficient of 35,219 M 1 cm 1 at 280 nm (20). Before
incubation, the solution was filtered through a 0.2 mm filter into sterile test
tubes. Samples were incubated at a specified temperature in a refluxed
reactor. Samples were taken out at intervals and stored on ice before addition
of CR or ThT.
Seeding solutions
To test whether seeding with preformed aggregates increases the rate of
HSA aggregation under the different conditions in which fibrils are formed,
a protein solution was incubated for 24 h and an aliquot that corresponded to
10% (w/w) of the total protein concentration was then added to a fresh
protein solution.
CR binding
Changes in the absorbance of CR dye produced by binding onto HSA were
measured in an ultraviolet-visible spectrophotometer (DU series 640; Beckman
Coulter, Fullerton, CA) operating at 190–1100 nm. All measurements
were made in the wavelength range of 220–500 nm in matched quartz
cuvettes. Protein solutions were diluted 20- to 200-fold into a buffer solution
with 5 mM of CR (Acros Organics, Geel, Belgium). Spectra in the presence
of the dye were compared with those of the buffer containing CR in the
absence of protein and also with those corresponding to the protein solution
without dye.
ThT spectroscopy
Protein and ThT were dissolved in the proper buffer at a final protein/dye
molar ratio of 50:1. Samples were continuously stirred during measurements.
Fluorescence was measured in a Cary Eclipse fluorescence spectrophotometer
equipped with a temperature control device and a multicell sample holder
(Varian Instruments, Palo Alto, CA). Excitation and emission wavelengths
were 450 and 482 nm, respectively. All intensities were background-corrected
for the ThT fluorescence in the respective solvent without the protein.
Protein fluorescence
To examine the conformational variations around the Tryp residue of HSA,
fluorescence emission spectra were recorded with a Cary Eclipse fluorescence
spectrophotometer equipped with a temperature control device and
a multicell sample holder (Varian Instruments). HSA samples were excited
at 295 nm, which provides no excitation of tyrosine residues and, therefore
does not cause emission or energy transfer to the lone side chain. Slit widths
were typically 5 nm.
Circular dichroism
Far- and near-ultraviolet (UV) circular dichroism (CD) spectra were
obtained using a JASCO-715 automatic recording spectropolarimeter (Jasco,
Tokyo, Japan) with a JASCO PTC-343 Peltier-type thermostated cell holder.
Quartz cuvettes with 0.2 cm pathlength were used. CD spectra were obtained
from aliquots withdrawn from the aggregation mixtures at the indicated
conditions and recorded between 195 and 300 nm at 25 C. The mean residue
ellipticity q (deg cm 2 dmol 1 ) was calculated from the formula:
148

Fibrillation Pathway of HSA 2355
q ¼ðqobs=10ÞðMRM=lcÞ, where qobs is the observed ellipticity in deg, MRM
is the mean residue molecular mass, l is the optical pathlength
(in centimeters), and c is the protein concentration (in g mL 1 ). To calculate
the composition of the secondary structure of the protein, SELCON3, CON-
TIN, and DSST programs were used to analyze far-UV CD spectra. Final
results were assumed when data generated from all programs showed
convergence (21).
XRD
XRD experiments were carried out using a Siemens D5005 rotating anode
x-ray generator. Twin Göbel mirrors were used to produce a well-collimated
beam of CuKa radiation (l ¼ 1.5418 A). Samples were put into capillary
with a diameter of 0.5 mm. X-ray diffraction patterns were recorded with
an imaging plate detector (AXS F.Nr. J2-394).
Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectra of HSA in aqueous solutions were
determined by using an FTIR spectrometer (model IFS-66v; Bruker) equipped
with a horizontal ZnS ATR accessory. The spectra were obtained at
a resolution of 2 cm 1 and generally 200 scans were accumulated to obtain
a reasonable signal/noise ratio. Solvent spectra were also examined under
the same accessory and instrument conditions. Each different sample spectrum
was obtained by digitally subtracting the solvent spectrum from the
corresponding sample spectrum. Each sample solution was repeated three
times to ensure reproducibility and averaged to produce a single spectrum.
Transmission electron microscopy
For transmission electron microscopy (TEM), suspensions of HSA were
applied to carbon-coated copper grids, blotted, washed, negatively stained
with 2% (w/v) of phosphotungstic acid, air dried, and then examined with
a Phillips CM-12 transmission electron microscope operating at an accelerating
voltage of 120 kV. Samples were diluted 20- to 200-fold when necessary
before deposition on the grids.
Scanning electron microscopy
Suspensions of HSA were applied to glass-coated stainless-steel grids,
blotted, washed, air dried, and then examined with an LEO-435VP scanning
electron microscope (Leica Microsystems GmbH, Wetlar, Germany) operating
at an accelerating voltage of 30 kV. Samples were diluted 20- to
200-fold when necessary before deposition on the grids. Microanalysis of
the scanning electron microscopy (SEM) samples was also performed to
avoid the presence of impurities.
Atomic force microscopy
Atomic force microscopy (AFM) images were recorded in tapping mode
by using a multimode SPM microscope equipped with a Nanoscope IIIa
controller from Digital Instruments (Santa Barbara, CA). The microscope
was coupled to an AS-12 resp. E-scanner and an Extender Electronics Module
EX-II, which allows acquisition of phase images. The AFM probes were typically
silicon SPM sensors (NCHR Nanosensors, Neuchatel, Switzerland).
Immediately after incubation, the protein samples were diluted 20–400 times
onto freshly cleaved muscovite mica (Sigma) attached to a magnetic steel disc
that served as the sample holder. The abrupt dilution of the samples immediately
quenched the concentration-dependent aggregation process. The AFM
samples were dried on air or under nitrogen flow when required. Control
samples (freshly cleaved mica, and mica and buffer solution) were also investigated
with AFM to exclude possible artifacts. Height and phase-shift data
were collected in the trace and the respective retrace direction of the raster.
The scan rate was tuned proportionally to the area scanned and was kept
within the 0.35–2 Hz range.
RESULTS
It is generally accepted that amyloid formation usually is
a result of misfolded and partially unfolded states acting in
competition with the normal folding pathways (22–24).
The HSA molecule is known to undergo several well-organized
changes during its conformation, usually under nonphysiological
conditions, as follows:
1. The N-F transition between pH 5.0 and 3.5 involves the
unfolding and separation of domain III without significantly
affecting the rest of the protein molecule
(25,26). The F form is characterized by a dramatic
increase in viscosity, lower solubility, and a significant
loss of helical content.
2. The F-E transition occurs between pH 3.5 and 1.2, and is
accompanied by a further protein expansion with a loss
of the intradomain helices of domain I. In addition,
the E form involves an increase in protein intrinsic
viscosity and a rise in the hydrodynamic axial ratio from
~4 to 9 (27).
3. The N-B transition occurs between pH 7.0 and 9.0, with
a slight reduction in helical content affecting the two interdomain
helices and a small increase in sheet structure (28).
4. In the presence of denaturant agents, such as urea, HSA
shows a two-step, three-state transition with an intermediate
(I) characterized by unfolding of the domain III and
partial but significant loss of native conformation of
domain I (29).
Therefore, an easy way to obtain at least partially denaturated
states is to induce a temperature-induced or solventinduced
protein denaturation process through incubation.
Thus, HSA was subjected to conditions previously found to
be effective for protein aggregation, in particular those that
induce amyloid-like fibril formation (19,30). HSA solutions
were incubated at a concentration of 20 mg/mL in 0.01 M
sodium phosphate buffer, pH 7.4, or 0.01 M glycine buffer,
pH 3.0, in the presence of 0 or 50 mM of added NaCl at
25 Cor65 C for 15 days. We chose 65 C as the incubation
temperature because HSA temperature-induced denaturation
takes place through a two-state transition with a first melting
temperature, Tm, of ~56 C and a second Tm of ~62 C(31,32)
as a consequence of the sequential unfolding of the different
domains of the protein, in particular, the IIA and IIIA
subdomains. Moreover, as the pH becomes more acidic,
T m becomes lower. We confirmed these melting temperatures
under our solution conditions by fluorescence spectroscopy,
and the results were in close agreement with previously
reported data (see Table S1 in the Supporting Material).
Therefore, it can be inferred that aggregation is unfavorable
below 65 C because protein folding competes with and
suppresses amyloid formation. It is known that hydrogen
bonding is weakened as temperature rises, but the hydrophobic
interaction becomes strengthened (33). As the heating
process continues, some of the cooperative hydrogen bonds
Biophysical Journal 96(6) 2353–2370
149

2356 Juárez et al.
that stabilized helical structure begin to break and expose
hydrophobic groups to the solvent, partially unfolding the
protein structure, which favors aggregation.
Physiological conditions: kinetics and amyloid
self-assembly of HSA
The propensity of protein solutions to form amyloid-like
aggregates under physiological conditions was assessed by
means of ThT fluorescence and CR absorbance measurements.
These two dyes specifically bind to ordered b-sheet
aggregates and, notably, to amyloid fibrils (34,35). Both
assays are necessary because positive ThT binding sometimes
does not occur with certain amyloid fibril systems (36).
Before incubation, native HSA does not display a capacity
to bind ThT. When incubated at physiological pH and room
temperature, the fluorescence emission intensity for the
protein at 482 nm is negligible, suggesting that HSA does
not form amyloid fibrils or other types of fluorescent aggregates
under these conditions. When the temperature is raised
to 65 C, a time-dependent increase in fluorescence is
observed (Fig. 1, a and b). The kinetics of HSA involved
the continuous rising of ThT fluorescence during the early
periods of the incubation procedure, and exhibited no discernible
lag phase until a quasi-plateau region was attained in the
FIGURE 1 Time evolution of ThT fluorescence in HSA solutions incubated
at 65 C at pH 7.4 in the (a) absence and (b) presence of 50 mM NaCl.
Biophysical Journal 96(6) 2353–2370
timescale analyzed. The addition of electrolyte favors a faster
formation of amyloid fibrils as a consequence of the screening
of electrostatic repulsions between protein molecules (37). In
fact, a gel phase can be observed after 4 days of incubation,
which denotes an enhanced development of fibril formation
because cross-links can be formed more easily, resulting in
a lower critical percolation concentration.
ThT fluorescence curves were fitted by means of nonlinear
square curve-fitting to a stretched exponential function
F ¼ F N þ DFexpð ½ksptŠ n Þ to obtain information on the
kinetics of amyloid formation. F, F N , and DF are the
observed fluorescence intensity at time t, the final fluorescence
intensity, and the fluorescence amplitude, respectively,
and k sp is the rate of spontaneous fibril formation. Although
the interpretation of the parameters involved in this equation
is not straightforward, these are useful for empiric descriptions
of the complex reactions whose kinetics is not easily
modeled (38,39). Values of ksp, n, and DF determined in
this way under the different conditions are shown in Table 1.
Values of n < 1 indicated that the kinetics can be approximated
to several exponential functions indicative of the
existence of multiple events in the amyloid formation.
Furthermore, larger ksp values in the presence of electrolyte
corroborate the more-efficient aggregation due to electrostatic
screening under high ionic strength conditions.
CR absorption also corroborates the formation of amyloid
fibrils, displaying a progressive red shift from 495 to ~530 nm
of the differential absorption maximum at 65 C in both the
absence and presence of added electrolyte, a typical feature
of amyloid fibers (35). Since absorption of HSA alone after
incubation contributes to only ~15% of the increase in CR
absorption, the change in absorption is mainly caused by
the formation of CR-binding species (see Fig. S1 in the Supporting
Material).
Fibrillation is independent of seeding
at physiological pH
Typical fibrillation processes involve a lag phase followed by
a relatively rapid elongation phase that stabilizes when all
TABLE 1 Kinetic parameters of the self-assembly process of
HSA solutions
F N
DF n ksp (h 1 )
pH 7.4
65 C
0 mM NaCl 85 83 0.56 0.019
50 mM NaCl 85 83 0.82 0.064
pH 3.0
25 C
0 mM NaCl 10 8 1.34 0.007
50 mM Na Cl 13 11 1.57 0.007
65 C
0 mM NaCl 8/30 6/22 1.25/3.6 0.144/0.005
50 mM NaCl 59 57 0.96 0.010
150

Fibrillation Pathway of HSA 2357
monomers have been incorporated into fibrils (40–42). In our
case, the absence of a lag phase at pH 7.4 would suggest that
nuclei are either formed very rapidly or the aggregation
process we monitor is not a classical nucleation-based fibrillation.
If nuclei consist of more than one molecule, their
formation will be reduced if the HSA concentration is lowered.
Nevertheless, when the protein concentration was
decreased to 0.5 and 2 mg/mL at 65 C, we did not observe
the appearance of a lag phase (figure not shown). In addition,
another feature that confirms a continuous fibrillation process
without a nucleation step is the absence of any remarkable
effect on the fluorescence curves when protein seeds were
added to protein solutions followed by subsequent incubation
(see Fig. S2). The aggregation rate during the growth phase
was unchanged by the addition of preformed aggregates and
followed apparent first-order kinetics. These characteristics
suggest that aggregation does not require nucleation, i.e.,
each protein monomer association step is bimolecular and
effectively irreversible, and there is no energy barrier to aggregate
growth. As discussed in detail below, TEM pictures
showed the formation of spherical oligomers after very short
incubation times. This usually occurs by a mechanism of classical
coagulation, or downhill polymerization (43), that does
not require a nucleation step.
Structural changes upon aggregation
at physiological conditions: secondary structure
To gain insight into the structural protein modifications upon
formation of amyloid-like aggregates, we recorded CD, FTIR,
and tryptophan (Tryp) fluorescence spectra. As a supplement
to ThT and CR assays, which provide information only about
the formation of fibrillar protein assemblies, far-UV CD and
FTIR data reveal the overall protein secondary structural
composition and their evolution to form amyloid-like or amorphous
aggregates. On the other hand, near-UV CD and Tryp
fluorescence data denote changes in protein tertiary structure
(for the latter technique, particularly in domain II of HSA).
Fig. 2, a and b, show far-UV CD spectra of HSA at pH 7.4
at 25 C and 65 C. The spectra at room temperature in the
absence of added salt show two minima—one at 208 and
other at 222 nm—characteristic of helical structure, which
remains unmodified upon incubation. When electrolyte is
present, a small decrease in ellipticity, [q], occurs as a consequence
of small changes in protein structure originating from
the formation of some amorphous aggregates in solution, as
shown in Fig. 2 a. In contrast, when the temperature is raised
to 65 C, the minimum at 222 nm progressively disappears
and [q] at 208 nm also strongly reduces (Fig. 2 b). This indicates
that high-temperature conditions spawn intermediates
that are clearly less helical than the starting conformations.
This change in CD spectra suggests the increment of either
b-sheet or loop structures, as discussed in detail further
below. The intensity loss of the 222 nm band indicates that
the increase in random coil/b-sheet conformations is accom-
FIGURE 2 (a) Far-UV spectra of HSA solutions at 25 C in the presence
of 50 mM NaCl at pH 7.4 at 1), 0 h; 2), 24 h; 3), 48 h; 4), 100 h; 5), 200 6),
and 7), 250 h of incubation. (b) Far-UV spectra of HSA solutions at 65 Cin
the presence of 50 mM NaCl at 1), 0 h; 2), 12 h; 3), 24 h; and 4), 48 h of
incubation.
panied by a reduction in the a-helical content of the protein
structure. At longer incubation times in the absence of added
salt in solution (~72 h), the CD spectrum resembles that
typical of proteins with high proportions of b-sheet structure,
which are characterized by a minimum at ~215–220 nm and
small [q] values as a consequence of the presence of
amyloid-like aggregates in solution. On the other hand, no
significant changes in far-UV CD spectra are observed
when electrolyte is added to solutions, except that the characteristic
features of b-sheet structure are present at earlier incubation
times at elevated temperature. A larger decrease in [q]
is also detected as a consequence of the increased number and
size of scattering objects in solution, which agrees with the
formation of a fibrillar gel upon longer incubation times.
Fig. 3 depicts the temporal evolution of the secondary
structure composition as revealed by CD analysis (21). At
pH 7.4 and room temperature, the initial a-helix content
is ~59%, the b-sheet conformation is ~5%, the turn content
Biophysical Journal 96(6) 2353–2370
151

2358 Juárez et al.
is ~13%, and the remaining random coil content is ~23%, in
agreement with previous reports (25). In the presence of
added electrolyte, no significant alterations in the structure
compositions are observed upon incubation; the initial
a-helix content is slightly reduced to ~55%, in contrast to
a very small increase in turn and b-sheet conformations.
After ~150 h of incubation, an additional slight decrease in
a-helix is observed due to an enhancement of protein aggregation,
i.e., the formation of a certain amount of protein
aggregates is observed, which is a characteristic feature of
globular proteins under high ionic strength conditions.
On the other hand, protein conformational compositions at
65 C after incubation indicate an increase in b-sheet conformation
from ~5% to ~21% at the expense of the a-helix
content (which diminishes from ~59% to ~20%) in the
absence of electrolyte. This is also reflected by the decrease
in the CD signal and the shift of the spectral minimum from
208 nm to longer wavelengths, as noted above. Coil and turn
conformations also change through the incubation process,
with values of ~27–34% and ~17–21%, respectively. The
alteration in structure composition seems to be stronger
when salt is added, due to electrostatic screening, which
favors protein disruption and association by modifying the
balance of interactions, with final b-sheet and a-helix
contents of ~26% and ~19%, respectively (Fig. 3). Moreover,
changes in secondary structure take place during the
first part of the incubation process in both the absence
(~90 h) and presence of electrolyte (~50 h), respectively,
and occur more rapidly under the latter condition. At very
long incubation times (>150 h) under high ionic strength
conditions, the increased scattering from the fibrillar aggregates
makes it difficult to estimate the secondary structure;
thus, these results are not shown in Fig. 3.
FTIR spectra were recorded at the beginning and end of
the incubation process by monitoring the observed changes
Biophysical Journal 96(6) 2353–2370
FIGURE 3 Time evolution of secondary structure compositions
of HSA solutions at pH 7.4 at 25 C(a and b)or65 C
(c and d) in the absence and presence of 50 mM NaCl, respectively.
(:) a-helix, (-) b-turn, (C) unordered, and (;)
b-sheet conformations.
in the shape and frequency of the amide I and II bands, and
the results corroborated the CD data. Before incubation, two
major absorption peaks in the spectral region of interest were
observed: the amide I band at 1653 (1652) cm 1 and the amide
II band at 1542 (1544) cm 1 in both the original and second
derivative IR spectra, respectively. This indicates the predominant
structural contribution of major a-helix and minor
random coil structures, in agreement with CD data (44–46).
For the amide I band (Fig. 4 a), a shoulder at ~1630 cm 1
can be also observed in the second derivative spectra that is
related to intramolecular b-sheet structure. Additional peaks
at ~1689 and 1514 cm 1 would correspond to b-turn and tyrosine
absorption, respectively (45). All of these peaks were also
observed in the presence of electrolyte at room temperature as
FIGURE 4 Second derivative of FTIR spectra at pH 7.4 of (a) native HSA
at 25 C before incubation, (b) HSA at 25 C in the presence of 50 mM NaCl,
and (c) HSA at 65 C in the absence of electrolyte.
152

Fibrillation Pathway of HSA 2359
incubation proceeded (Fig. 4 b), compatible with little changes
in secondary structure composition, as noted above.
After incubation at 65 C, a red shift of the amide I band
from 1652 to 1658 cm 1 and a blue shift of the amide II
band to 1542 cm 1 in the second derivative spectrum are
indicative of a certain increase of disordered structure, as
revealed by far-UV CD (Fig. 4 c). The appearance of
a well-defined peak around 1628 cm 1 (1626 cm 1 in the
presence of added salt) points to a structural transformation
from an intramolecular hydrogen-bonded b-sheet to an intermolecular
hydrogen-bonded-b-sheet structure (47). The spectrum
also shows a high-frequency component (~1692 cm 1 )
that would suggest the presence of an antiparallel b-sheet
(48). In addition, a little shoulder around 1534 cm 1 also
was assigned to b-sheet after incubation at high temperature
(49). The increase in the band associated with b-sheet in the
FTIR spectra correlates well with the large changes in CD
spectra. On the other hand, peak shifts are slightly more abrupt
when electrolyte is present in solution because the aggregation
is stronger (plot not shown). This leads to the formation
of a fibrillar gel favored by the decrease in electrostatic repulsions
and a change in the hydration layer surrounding the
protein molecules that allows interfibrillar attachment.
Structural changes upon aggregation at
physiological conditions: tertiary structure
At room temperature, the near-UV CD spectrum showed two
minima at 262 and 268 nm and two shoulders around 275 and
285 nm, characteristic of disulphide and aromatic chromophores
and the asymmetric environment of the latter (Fig. 5
a)(50). These features are significantly retained during incubation
in the presence of electrolyte excess despite the formation
of some amorphous aggregates in solution, as noted
above (figure not shown). On the other hand, when the incubation
temperature is raised to 65 C, important alterations in
the near-UV CD spectra occur in both the absence and presence
of electrolyte: ellipticity decreases, and the minima at
262 and 268 nm progressively disappear as incubation
proceeds. In addition, a significant loss of fine structure
detectable in the region of 270–295 nm also occurs. Nevertheless,
these changes in near-UV CD data are less marked
than in far-UV measurements because secondary structural
changes are more sensitive to temperature than tertiary structural
changes (51). These effects corroborate alterations in
tertiary structure upon fibrillation conditions.
The behavior described by near-CD UV measurements is
also supported by tryptophanyl fluorescence data. HSA has
a single tryptophanyl residue, Tryp 214 , located in domain
II. A very small change in fluorescence emission occurs
with incubation at room temperature in the presence of electrolyte,
confirming the existence of little variations in tertiary
structure, in agreement with previous data (not shown). In
contrast, when the temperature is raised to 65 C, a 4 nm
hypsochromic shift (from 341 to 337 nm) takes place accom-
FIGURE 5 (a) Near-UV CD spectra of HSA solutions at pH 7.4 and 65 C
in the absence of electrolyte at 1), 0 h; 2), 6 h; 3), 12 h; 4), 24 h; and 5), 48 h.
(b) Time evolution of Trp fluorescence of HSA solutions at 65 C in the (-)
absence and (B) presence of 50 mM NaCl.
panied by a reduction in fluorescence intensity: the domain II
of HSA unfolds in such a way that the Trp 214 residue of HSA
located at the bottom of a 12 A˚ deep crevice (52) finds itself
in a more hydrophobic environment (32). As incubation at
elevated temperature proceeds, conversion of protein molecules
to fibrils is accompanied by an additional burial of
the Tryp residue, as indicated by the decrease of the Tryp
fluorescence emission intensity (Fig. 5 b) and the further
blue shift of the emission maximum from 337 to 333 nm
(53). All of this points to the strong involvement of tertiary
structure changes of domain II of HSA in the fibrillation
mechanism. This change is enhanced and occurs more
rapidly in the presence of added electrolyte, which confirms
a larger structure alteration as a consequence of enhanced
intermolecular interactions to give fibrillar assemblies. This
also agrees with the increasing content of b-sheet conformation
revealed by CD and FTIR measurements.
In situ observation of fibrillar structures at
physiological pH: TEM
TEM pictures were recorded at different stages of the incubation
period for each of the conditions described above.
Distinct time-dependent morphological stages can be
Biophysical Journal 96(6) 2353–2370
153

2360 Juárez et al.
observed in these images. Thus, at room temperature neither
fibrils nor other types of aggregates are detected, except for
small amorphous protein clusters observed in the presence of
electrolyte after a long incubation period (150 h), which
possess a largely helical structure (Fig. 6 a). In contrast,
when the temperature is raised to 65 C, fibril formation is
observed. Electron microscopy indicates that aggregation
leads first to a globular species that subsequently converts
to fibrils with a curly morphology. The fibrillation pathway
in the presence of electrolyte is very similar to that observed
in its absence but it takes place in a shorter timescale, in
agreement with previous results. Fig. 6, b–j, show electron
micrographs of the sample heated at 65 C at different steps
of the incubation period in the presence of 50 mM of electrolyte.
The number and length of the fibrils has increased in
relation to other structures, although several morphologies
can be observed throughout incubation.
Small spherical clusters of ~20 nm formed by protein oligomers
are observed (Fig. 6 b) at short incubation times (5 h).
These aggregates present relatively few changes in their
tertiary and secondary structures, as shown by CD and fluorescence
data. With further incubation (Fig. 6 c, 15 h), a certain
elongation of these spherical aggregates can be observed. This
bead-like structure at short incubation times arises from what
appears to be attractive interactions between spherical
a b
c
e f
d-1) d-2)
proteins aggregates, as shown in Fig. 6 d-1 (see also
Fig. S3), which may result in an increased exposure of hydrophobic
residues, giving rise to more elongated structures. This
elongation involves a conformational conversion of protein
structure to consolidate the structure, and in all probability
it implies changes in the hydrogen-bonding status (Fig. 6
d-2). This is in agreement with a further development in
ThT fluorescence and decreases in both helical content and
Tryp fluorescence at this incubation point, as shown previously.
On the other hand, we did not find evidence of
formation of elongated structures by longitudinal fusion of
oligomers, as recently reported (54).
Bead-like structures progressively become more elongated
upon incubation (35 h) due to mutual interactions
between these structures and subsequent annealing, and
convert into short protofibrils (Fig. 6 e), in agreement with
a decrease in helical structure as revealed by CD. Alterations
in the conformational structure of these oligomers and subsequent
elongation via monomer addition may also be present;
however, the TEM resolution did not allow us to confirm
that. Several authors reported a tendency for these beadlike
structures to transform into fibrillar structures at elevated
temperatures caused by partial unfolding of the protein molecules
and giving rise to conditions conductive to fibril formation
(55–57).
g h
i j
FIGURE 6 TEM pictures of the different stages of the HSA fibrillation process at pH 7.4: (a) at25 C in the presence of 50 mM NaCl after 150 h of incubation,
and at 65 C in the presence of 50 mM NaCl after (b) 5 h; (c) and (d) 15 h (part d shows the elongation of oligomers to give bead-like structures); (e)35h
(where short protofibrils are observed); (f)45h;(g) 50 h (where long curly fibrils are seen); and (h–k) after 72 h. Part i shows the addition of oligomers to mature
fibrils, j shows the association of mature fibrils in bundles, and k shows mature fibrils with ribbon-like structure.
Biophysical Journal 96(6) 2353–2370
k
154

Fibrillation Pathway of HSA 2361
Further incubation results in the presence of more fibrils,
which increase in length as incubation proceeds (Fig. 6,
f and g). After 2 days of incubation, numerous longer
curly-branched and interconnected fibrils are present
(Fig. 6 g), with lengths and widths characteristic of classical
amyloid fibrils (i.e., between 0.5 and several micrometers in
length and 9–10 nm in width (3,5,9)), as detected by TEM
and AFM (see Fig. 6 g and Fig. S4). The appearance of
this structure coincides well with the plateau region of ThT
binding, the far UV-CD profiles characterized by minima
around 215–220 nm, and the intermolecular b-sheet structure
observed by FTIR. Most fibrils appear curly and interconnected,
and some of them are even circular, as observed
previously for other proteins, such as b 2 microglobulin
(58) and a-crystallin (56).
If the incubation time is extended further (3 days), one can
observe mature straight fibrils, which can be seen as a structural
evolution of curly fibrils (Fig. 6 h). Mature fibrils are
thicker and stiffer than single fibrils and seem to be formed
by lateral (side-by-side) assembly of two or more individual
filaments. These mature fibrils are less numerous in the
absence of electrolyte because the electrostatic screening is
lower, avoiding direct contact between constitutive fibrils.
We have also observed that lateral interactions of single
particles collaborate in growing these straight mature fibrils
(57). Scrutiny of the protein aggregates indicates that HSA
particles and clusters tend to align within the immediate
vicinity of the fibers (Fig. 6 i), serving the single fiber as
a lateral template or scaffold for small protein molecules,
and would constitute a subcomponent in mature fibrils. It
is worth mentioning that some fibril solutions when analyzed
by SEM show the existence of very long fibers, exceeding
200 mm (see Fig. S5). We think that this effect may result
from the air-drying process favoring attractive interactions
between single fibers during the solvent removal.
In addition, a structural diversity of mature fibrils is noted:
flat ribbons are observed in solution, as well as long, straight
fibril ensembles (Fig. 6 j). This polymorphism may arise
from variations in the quaternary structure, the manner in
which protofilaments self-associate, or the protofilament
substructure (e.g., in the details of hydrogen-bonding
networks and side-chain packing) (59). Finally, one of the
characteristic traits of the mature amyloid fibrils is their
tendency to bend, twist, and agglomerate. Fig. 6 k shows
laterally connected fibers that split over a certain distance
or overlap each other. The extent and rate of this growth is
dependent on solution conditions and lateral interactions
between fibrils, which are responsible for the thickening of
the mature fibrils and the formation of suprafibrillar aggregates.
Thus, we conclude that pseudo-globular aggregates
rearrange slowly to form linear, curly fibrils. These may be
sufficient to produce a high-affinity template that is subsequently
elongated by monomeric units or other fibrils, and
can lead to the formation of ordered, straight, or ribbonlike
fibrillar structures.
Fibril structure: XRD
The amyloid-like character of the fibrillar aggregates detected
by TEM was confirmed by XRD. The XRD image of the HSA
fibrils is shown in Fig. 7. Two strong reflections can be
observed: a dominant sharp and intense reflection occurs at
4.8 A˚ , and one weaker, more diffuse, but still intense reflection
is observed at ~11 A˚ . The 4.8 A˚ meridional reflection
arises from the spacing between hydrogen-bonded individual
strands in the b-sheet structure that lie perpendicular to the
fibril axis, and the 11 A˚ equatorial reflection corresponds to
the intersheet spacing, with the b-sheets stacked face to face
to form the core structure of protofilaments (60,61). This indicates
that fibrils possess a cross-b structure, one of the diagnostic
hallmarks of amyloid structures.
Acidic conditions: kinetics and amyloid
self-assembly of HSA
We next subjected HSA to acidic pH and assessed its propensity
to form amyloid fibrils under such conditions in both the
presence and absence of 50 mM NaCl. Increases in ThT fluorescence
were observed at both 25 C and 65 C, although the
increase was quite small at 25 C. This indicates the formation
of a small amount of additional b-sheet conformation as
a consequence of the formation of oligomeric aggregates, as
will be shown below (Fig. 8). At 65 C, the increase in
ThT fluorescence in the absence of electrolyte is lower than
that obtained at physiological pH and extends over a larger
period of time. This can be a result, on the one hand, of a lower
capacity of fibrillation under these conditions (see below) or,
on the other, to the presence of a lag phase if compared with
solution to which electrolyte is added, as discussed further
below. After a small increase in ThT fluorescence at relatively
short incubation times, a plateau occurs at ~24–100 h, after
which the ThT fluorescence starts to increase again. Thus, it
FIGURE 7 XRD pattern of HSA fibrils.
Biophysical Journal 96(6) 2353–2370
155

2362 Juárez et al.
is thought that under acidic conditions in the absence of electrolyte,
oligomeric structures (protein clusters) are formed in
a series of thermodynamically unfavorable assembly steps
followed by a growth phase in which clusters are elongated
by further addition of protein monomers and/or oligomers
upon mutual interaction.
Fitting of the time ThT fluorescence evolution shows us
that under acidic conditions, the self-assembly process
becomes more cooperative, as indicated by the values of
n > 1. This indicates the different nature of the self-assembly
pathway of HSA under acidic conditions with respect to
physiological pH, since interactions between protein molecules
are modulated by changes in both the pH and the initial
protein structure (62). In addition, the plot in the absence of
electrolyte is fitted in two steps: 1), the fast formation of
small clusters; and 2), the lag phase, which can originate
from the necessity to reach a critical concentration of clusters
for aggregation to continue. Probably, oligomeric species
formed in very early stages of the aggregation process
(whose existence is indicated by the slight increase of ThT
fluorescence at very short incubation times) are more soluble
under acidic conditions, and only after they achieve a critical
concentration are they able to grow to generate larger aggregates
(63). However, complete fibril formation can be
achieved only in the presence of electrolyte.
Nucleation-dependent growth mechanism
in acidic medium
To corroborate the origin of the plateau region in the absence
of electrolyte at elevated temperature, we performed a seeding
fibril growth under the conditions previously specified. When
seeds are added to the protein solution, a continuous increase
in ThT fluorescence is observed. This is the typical behavior
observed for a nucleation-type growth mechanism and corroborates
the existence of this lag phase (see Fig. S6). On the other
hand, no changes are observed when protein seeds are added
Biophysical Journal 96(6) 2353–2370
FIGURE 8 Time evolution of ThT fluorescence in HSA
solutions incubated at pH 3.0 at 25 C(a and b) or65C
(c and d) in the absence and presence of 50 mM NaCl,
respectively.
to a solution containing 50 mM NaCl. The difference between
both solution conditions may arise from the greater hydrophobicity
of oligomeric species formed during very earlier incubation
stages in the presence of excess electrolyte. In its absence,
electrostatic repulsion between oligomeric species seems to
preclude for some time the formation of nuclei with a critical
size to overcome the energy barrier that impedes aggregation.
Structural changes at acidic pH: secondary
structure
At acidic pH and room temperature, both minima in [q] at 208
and 222 nm are still present before incubation proceeds, which
indicates an important retention of this type of structure, as
previously described (25,64). During incubation, a slight
decrease in [q] is observed in both the absence and presence
of added electrolyte, which is compatible with a small
decrease in a-helices and the development of a small amount
of b-sheet conformation due to the appearance of small amorphous
aggregates in solution (figure not shown). In contrast,
a shift of the 208 nm minimum to lower wavelengths takes
place as incubation proceeds (0–200 h) at 65 C in the absence
of electrolyte (Fig. 9 a). An increase in random coil structure
(characterized by a single minimum below 200 nm) can
account for this shift. Upon further incubation, an additional
red shift occurs as a consequence of the increase of b-sheet
structure in the aggregates formed, as also indicated by the
increase in ThT fluorescence. The far-UV CD curves at
65 C in the presence of NaCl followed a trend similar to those
obtained at physiological pH, although the ellipticity decrease
is less severe at long incubation times as a consequence of
a lower amount of scattered light from fibril aggregates (see
Fig. 9 b). This confirms the lower fibrillar density under these
conditions, which also precludes the formation of a fibrillar
gel, in contrast to physiological conditions.
The secondary structure compositions in acidic solution at
room temperature at the beginning of the incubation process
156

Fibrillation Pathway of HSA 2363
FIGURE 9 Far-UV spectra of HSA solutions at 65 C at pH 3.0 in (a) the
absence of electrolyte at 1), 0 h; 2), 24 h; 3), 125 h; 4), 175 h; 5), 200 h;
and 6), 250 h of incubation; and (b) the presence of 50 mM NaCl at 1),
0 h; 2), 15 h; 3), 48 h; 4), 100 h; 5), 150 h; and 6), 200 h of incubation.
indicate a reduction in a-helix conformation (~45%) and an
increase in b-sheet, b-upturn, and coil contents (7%, 20%,
and 28%, respectively), typical of the acid-expanded E-state
of HSA (25) (Fig. 10). No significant changes were detected
in structural composition during the incubation procedure at
room temperature in either the absence or presence of added
electrolyte up to 100 h incubation. At this stage, a slight
increase in b-sheet and unordered conformations is observed
for both solution conditions. In contrast, when the temperature
is raised to 65 C, an important increase in b-sheets at
the expense of helical conformation occurs as also observed
at physiological pH. This is also accompanied by an increase
in unordered conformation at early incubation times (0–150
h). The change in secondary structure occurs during a longer
time interval (up to 9 days) in acidic solution. The final ahelix
and b-sheet compositions are respectively 24% and
16% in the absence of electrolyte (17% and 21% in the presence
of 50 mM NaCl), compared to 20% and 21% at neutral
pH. This indicates small compositional changes in the resulting
amyloid aggregates. The turn conformation also shows
little changes throughout incubation.
FTIR experiments corroborate the structural changes
undergone by the protein molecules as incubation proceeds
at acidic conditions. Before incubation, the amide I band
at 1650 cm 1 and the amide II band at 1542 cm 1 confirm
that there is still a significant amount of a-helices. Incubation
at room temperature under acidic conditions leads to a certain
increase of the peak located at ~1627 nm, which corresponds
to intramolecular b-sheet structure, in agreement with
ThT fluorescence and CD data. On the other hand, after
incubation at 65 C, a red shift of the amide I band from
1650 to 1656 cm 1 and a blue shift of the amide II band to
1540 cm 1 are indicative of an increased amount of disordered
structure. In addition, the shift and further increase of
the 1627 cm 1 peak to 1624 cm 1 also points to a structural
transformation from an intramolecular hydrogen-bonded
b-sheet to an intermolecular hydrogen-bonded b-sheet structure
(46), as seen for physiological pH. Spectra also show
a small high-frequency component (~1692 cm 1 ) that would
suggest the presence of antiparallel b-sheet (47) (see Fig. S7).
Structural changes at acidic pH: tertiary structure
When the pH is decreased, there is an increase in [q]between
260 and 280 nm, and a slight decrease between 285 and
300 nm, denoting loss of tertiary structure. Nevertheless, there
are still significant CD signals left, suggesting a remaining
tertiary structure, in agreement with previous reports (25,65).
These changes at acidic pH are related to structural rearrangements
of all HSA domains. In particular, some increase in ellipticity
below 295 nm takes place during incubation at room
temperature, which points to little further tertiary structural
changes as aggregation takes place; in particular, a loss of
fine structure is detectable in the 270–295 nm region (see
Fig. S8 a). At 65 C, changes in tertiary structure are more
important; in particular, an almost complete absence of the
minima is observed between 260 and 270 nm, indicating
a further loss of asymmetry around disulfide bridges and/or
aromatic residues as incubation proceeds. An additional loss
of fine structure in the range of 280–295 nm, similar to that
observed at physiological pH, is also detected (Fig. S8, b and c).
Upon incubation at acidic pH and room temperature,
a certain decrease in the tryptophanyl fluorescence and a slight
blue shift of the emission maximum occur between days 0 and
8 of incubation, which points to a certain internalization of
Tryp to the nonpolar environment of domain II as a result of
certain aggregation under these conditions (see Fig. S9).
Because changes in far- and near-UV CD spectra are relatively
small for this incubation period, this leads us to think that
structural changes in domain II of HSA are involved in this
aggregation process (66). At 65 C, the tryptophanyl residues
are in a more solvent-exposed environment during the incubation
because the fluorescence intensity abruptly decreases
Biophysical Journal 96(6) 2353–2370
157

2364 Juárez et al.
during the first 4 days of incubation as a result of the sequential
unfolding of domains I and II of HSA. This period of time is
slightly shorter than at pH 7.4 (mainly in the presence of electrolyte
excess) because the expanded E state already involves
an important alteration in the tertiary structure.
In situ observation of fibrillar structures at acidic
pH: TEM
HSA samples obtained at different incubation times were
also subjected to TEM analysis under acidic conditions. At
room temperature in the absence of salt, no aggregates
Biophysical Journal 96(6) 2353–2370
FIGURE 10 Time evolution of secondary structure
compositions of HSA solutions at pH 3.0 at 25 C(a and
b) or65C(c and d) in the absence and the presence of
50 mM NaCl, respectively. (:) a-helix, (-) b-turn,
(C) unordered, and (;) b-sheet conformations.
were observed during the first 2 days of the incubation
process. From the third day, the presence of small clusters
(globules) of aggregated protein could be observed. These
aggregates have a globular or spherical shape, not the regular
fibrillar appearance associated with amyloid structures (see
Fig. 11 a). In addition, less numerous, more elongated aggregates
(20–30 nm long, 3–4 nm wide) can be also observed,
and their population slightly increases as incubation
proceeds. The formation of these types of aggregates is characterized
by a decrease of helical structure accompanied by
a slight rise in b-sheet and unordered conformations from
CD measurements at long incubation times.
FIGURE 11 TEM pictures of the different stages of the
HSA fibrillation process at pH 3.0 in the presence of 50
mM NaCl (a) at25C after 150 h of incubation, and at
65 C after (b) 24h,(c) 150 h, and (d) 250 h of incubation
in the presence of 50 mM NaCl.
158

Fibrillation Pathway of HSA 2365
When the temperature is raised to 65 C, fibril formation
takes place in several steps. First, formation of quasi-spherical
aggregates takes place under incubation times similar to
those at pH 7.4, and these seem to be fairly soluble. On the
other hand, we have found that a coexistence of spherical
aggregates with circular ring-shaped particles of larger size
(~100–300 nm) is observed at short incubation times (on
the order of a few hours) in the presence of excess electrolyte
(Fig. 11 b). These structures appear as a possible intermediate
structural rearrangement of smaller protein aggregates
before fibril formation facilitated by electrostatic screening.
There is no evidence as to whether structural reorganization
to form fibrils takes place within this type of aggregates, or
whether dissociation of HSA molecules from these structures
occurs. Based on our CD data, which indicate that a progressive
gaining of b-sheet structure and unordered conformation
at the expense of a-helices takes place in the first part of the
incubation process, we suggest that only after a certain critical
amount of b-sheet structure is reached inside these ringshaped
particles will short fibrils be formed in solution from
decomposition of the former and become stable. Once sufficient
molecules are present within the oligomer, reorganization
steps become thermodynamically favorable as a result of
an increase in the number of hydrogen bonds and other stabilizing
interactions. Once fragments of highly ordered aggregates
are present, the free energy for addition of monomeric
molecules to a growing fibril will become more favorable.
Similar circular ring-shaped structures have also been
observed as a structural intermediate before the formation
of amyloid fibrils in the self-assembly process of insulin
(67), Ab17-42 peptide (68), Ab1-4 peptide, and HaPrP23-
144 prion protein (69). In this case, the circular structures
may incorporate into fibrils but also self-aggregate to form
large, amorphous structures (57). Given the large structural
differences between HSA insulin and Ab17-42 peptide, and
their distinct propensity to form amyloid fibrils, it seems
reasonable to think that these circular ring-shaped structures
can be a sort of common structural intermediate of amyloid
fibril formation under different solution conditions.
After longer incubation times (6 days) for HSA solutions in
the absence of electrolyte at 65 C, small, well-defined, short
protofibrils (~100 nm long and 3–4 nm wide) start to appear
(Fig. 11 c). The corresponding CD spectrum shows a progressive
increase in the proportion of b-structure at the expense of
the helical one during incubation. In contrast, the presence of
electrolyte involves an additional step: the formation of short
curly fibers occurs in a broader incubation timescale than at
physiological pH (Fig. 11 d); under further incubation, the
fibrils appear to increase slightly in number and length.
They range from ~100 nm to several hundred nanometers in
length and 8 to 11 nm in width; thus, they are shorter on
average than those obtained at physiological conditions but
have the same average width. Closed fibril loops with diameters
of ~100 nm were also frequently observed because the
formed filaments can remain short and thin to enable them
to bend and form closed rings, as also observed for b2 microglobulin
(58), a-crystallin (56), equine lysozyme (70), insulin
(67), and a-synuclein (71). This fibrillar material appears
similar to protofilaments observed in the early stages of other
amyloidogenic systems, given the small length and the
absence of higher-order structures.
DISCUSSION
The ability to form amyloid fibrils is a generic property of
polypeptide chains, although the propensity of different
regions of proteins to form such structures varies substantially
(72). The properties of unfolded polypeptides, including their
relative propensities for a- and b-structure, their intrinsic
solubility, and the nature of the interactions within the resultant
fibrillar structures, are likely to be particularly important
determinants in their relative abilities to form fibrils. The
conformation of the partially folded state is not by itself a critical
feature of fibril formation; rather, it is suggested that the
basis for amyloidogenesis is the presence of partially denaturation
conditions that destabilize the native fold of the protein
but do not preclude noncovalent interactions between the
various groups within the protein. In our case, it appears
necessary to destabilize HSA molecules and hence to generate
a partially folded intermediate that can aggregate to form
fibrils. Thus, one can readily atttain conditions in which
such aggregation-prone intermediate states are significantly
populated by lowering the pH or raising the temperature.
ThT fluorescence, CR staining, XRD, and TEM pictures
demonstrate the formation of amyloid-like structures under
these conditions. The aggregation process is governed by
the balance between attractive and repulsive interactions
between protein molecules. Conformational changes induced
by heat increase the number of hydrophobic residues exposed
to the aqueous solvent. The exposure of these groups results in
attractive hydrophobic interactions that play a dominant role
in the aggregation process. Repulsive forces are induced by
a surface charge that can be modulated by changes in pH,
which controls the net charge of the protein, and by the ionic
strength of the solvent, which controls the screening of electrostatic
interactions (73).
Absence of a lag phase in HSA fibrillation process
in most solution conditions
A first characteristic of the HSA fibrillation process is the
absence of a lag phase, as previously observed for bovine
serum albumin (BSA) (74) and acyl phosphatase (75) under
all solutions conditions analyzed except for acidic pH at
room temperature. This occurs if the initial aggregation is
a downhill process that does not require a highly organized
and unstable nucleus. This is supported by the fact that
seeding of preformed aggregates does not accelerate the
fibrillation process. In this regard, it has been suggested
that large multidomain proteins like BSA are able to form
Biophysical Journal 96(6) 2353–2370
159

2366 Juárez et al.
propagation-competent nucleus-like structures (oligomeric
structures) (74). In our case, TEM pictures also show the
formation of spherical oligomers upon very short incubation
times, which occur by a means of classical coagulation mechanism.
In contrast, the presence of a certain lag phase upon
incubation in acidic medium at 65 C in the absence of electrolyte
may well indicate that oligomeric aggregates need more
time to develop and/or persist for longer times because of their
enhanced solubility, so they need to reach a certain number or
size to change the energy landscape of the system and promote
further aggregation.
Existence of different intermediates on the HSA
fibrillation pathway
On a macroscopic scale, the different steps in the fibril formation
pattern, as observed by TEM, consist of the formation of
nonfibrillar aggregates (oligomeric globules) and their subsequent
elongation (bead-like structures and circular ringshaped
structures), and the development of protofilaments
and their assembly in fibrils, which can rearrange in more
complex structures. On a molecular level, CD and FTIR results
show that HSA possesses native-like a-helical characteristics
with residual b-sheet content before acidic or heat treatment.
In very early periods of incubation, HSA forms small,
soluble, globular oligomers of mainly native-like molecules
in acidic, physiological, and/or high-temperature conditions,
with a progressive increase in b-sheet content and unordered
conformation upon further incubation, as revealed by ThT
fluorescence and far-UV CD. Thus, upon further incubation
(5–50 h) at elevated temperature, the spectroscopic characteristics
indicate losses of persistent tertiary structure along with
unfolding of certain secondary structure to different extents
depending on the solution conditions (acidic or physiological
pH, added salt). Different conditions may cause different
regions of the polypeptide chains that are relatively flexible
and not involved in strong intramolecular interactions
(76,77) to enhance the aggregation process, leading to an
evolution of the previous globules into additional intermediate
structures (bead-like structures and circular ring-shaped structures
at physiological and acidic pH, respectively, with the
latter found only at high ionic strength). Bead-like structures
arise from what appears to be attractive interactions between
globular protein oligomeric clusters, which may result from
an increased exposure of hydrophobic residues, mainly in
the presence of electrolyte. We found no evidence of formation
of elongated structures by longitudinal fusion of oligomers.
These bead-like structures progressively become more elongated
upon incubation (35 h) due to mutual interactions
between them and convert into protofibrils, in agreement
with a decrease in helical structure as indicated by CD data.
The decrease in diameter accompanying elongation may be
explained on the basis of reorganization of structure, in particular
of the b-strands. Several authors have also reported the
tendency of these bead-like structures to transform into fibrillar
Biophysical Journal 96(6) 2353–2370
structures at elevated temperatures caused by partial unfolding
of the protein molecules and giving rise to conditions conducive
to fibril formation (55–57), as shown in Fig. 6 d.
The combination of experimental observations described
here indicates that the formation of fibrils from soluble HSA
molecules proceeds in a series of stages, the first of which is
effectively the presence of oligomeric globules. Thus, the
role of association-competent oligomeric intermediates may
result in a kinetic pathway via clustering of these oligomeric
species, which can be rationalized in the light of colloid coagulation
theory, i.e., the formation of a critical oligomer or an
ensemble of critical oligomers and subsequent aggregation
into bead-like structures, and then protofibrillar structures
(78). The persistence of these spherical oligomers in solution
coinciding with fibril assembly also supports the view that
they may be ‘‘on-pathway’’ intermediates (see Fig. 6 and
Fig. S10). Spherical oligomeric structures have been
proposed to serve a key, on-pathway role in both the formation
and elongation of amyloid fibrils of the Sup35 (79) and
Ure2p yeast prion proteins (80).
In contrast, we speculate that the circular ring-shaped structures
found in acidic medium at high ionic strength, which
appear to be composed of two semicircular units, may come
from bending and association of early-formed, short, beadlike
structures due to a decrease in their solubility. This
decrease would stem from the electrolyte concentration
present in solution, which screens electrostatic interactions
between aggregates. Once sufficient molecules are present
within this type of intermediate structure, reorganization steps
become thermodynamically favorable as a result of an increase
in the number of hydrogen bonds and other stabilizing interactions.
Once critical fragments of ordered aggregates are
present (with a critical amount of b-sheet structure), the free
energy for addition of monomeric molecules to a growing
elongated structure becomes more favorable. In this way, short
protofibrils will be formed in solution upon dissolution of the
ring-shaped intermediate structures and will become stable.
These intermediates are not observed by TEM once protofibrils
start to be observed, which suggests that they may act
as reservoirs of the initially very short protofibrils. However,
a deeper structural analysis of this structural intermediate
and its evolution to protofibrils is needed, and is currently
under way. On the other hand, the fact that some elongated
structures (but not fibrils) are formed in acidic pH at room
temperature, in contrast to physiological medium, for which
no aggregation is observed except for some amorphous aggregates,
suggests that the charge distribution on the protein influences
the propensity to form amyloid fibrils. The neutralization
of negative charges at acidic pH may promote this elongation.
HSA fibrils are curly
The thermodynamic instability of prefibrillar aggregates
causes them to evolve into more stable assemblies upon additional
incubation (>75 h), eventually leading to the appearance
160

Fibrillation Pathway of HSA 2367
of stable fibrils, mainly at physiological conditions at elevated
temperatures, where structural reorganization results in the
large majority of hydrophobic groups being concealed from
the solvent. This is corroborated by ThT fluorescence, far
UV-CD, and FTIR spectra, which demonstrate that the major
elements of ordered secondary structure are b-sheets, suggesting
that the a-helical regions of the native protein have undergone
significant structural changes. TEM pictures corroborate
this picture, showing that the fibrils formed are mainly, narrow,
branched, and elongated but curly, in contrast to the straight
needle-like structures characteristic of bona fide fibrils. As
incubation proceeds, these worm-like structures increase in
length. These fibrils display the typical 4.8 A˚ peak indicating
the typical interstrand distance of classical fibrils, and the
11 A˚ equatorial reflection corresponds to the intersheet
spacing, with the b-sheets stacked face to face to form the
core structure of protofilaments (81). Based on the CD and
FTIR data, we speculate that these types of fibrils may be
formed by a seam of b-sheet structure decorated by relatively
disorganized a-helical structure, as previously observed for
RNase A (82) or yeast Ure2p (83). In addition, an antiparallel
arrangement of b-strands forming the b-sheet structure of the
HSA fibrils seems to be the most probable configuration, as
denoted by the band at ~1691–1693 cm 1 in the FTIR spectra.
Curly aggregates have been also seen in other proteins, such as
b2 microglobulin (58) and a-crystallin (56). In addition, intramolecular
end-to-end association of short individual filaments
appears to be favorable, as TEM images reveal the presence of
a substantial number of closed loops appearing to form spontaneously,
mainly in the absence of electrolyte at acidic conditions.
Qualitatively, the probability of a single fibril joining
end-to-end to form a closed loop will be high if the fibril is
sufficiently flexible and of appropriate length for the ends to
find themselves regularly in close proximity to each other
(84). Indeed, the results presented here exemplify the favorable
nature of loop formation when such fibril morphologies are
adopted. Loop formation was reported previously for other
amyloid-forming systems (85,86).
On the other hand, it is interesting to note that a similar
curly morphology for HSA can be achieved under different
solution conditions: the single filament, in the form of both
open flexible chains and closed loops, is observed at both
physiological and acidic pH in the presence of electrolyte.
Nevertheless, the rate and extent of aggregation depends
on the solution conditions: the amount of formed fibrils
and their length (larger at physiological conditions) favor
interactions between early aggregates to form fibrils. This
is revealed by the formation of a gel phase under suitable
solution conditions.
Curly fibers can evolve into a suprafibrillar
structure
Very long incubation times (>150 h) lead to a more complex
morphological variability among amyloid fibrils (e.g., long
straight fibrils, flat-ribbon structures, or laterally connected
fibers). These compact, mature fibrillar assemblies formed
at the endpoint of the aggregation process may result from
an effort to minimize the exposure of hydrophobic residues,
and are also likely to result in increased van der Waals interactions,
leading to greater stability, as previously noted for
b2-microglobulin (87), a-synuclein (5), and insulin (88). A
similar progression in structure from curly fibrils to mature
straight fibers was also observed for Ab-peptide (89) and
insulin (90). In contrast, BSA fibrillation was shown to be
halted at the early curly stage, despite the enormous structural
similarities with HSA, since no further development
in fibrillar structure over long timescales was observed
(74). Mature fibrils are thicker and stiffer than single fibrils
and appear to be formed by lateral (side-by-side) assembly
of two or more individual filaments. Nevertheless, under
physiological conditions, we observed that mature straight
fibrils can also grow by swollen protein particles tending
to align within the immediate vicinity of the fibers, as shown
in Fig. 6 i, serving the single fiber as a lateral template or
scaffold for small protein molecules, and would constitute
a subcomponent in mature fibrils. Their population is dependent
on solution conditions and lateral interactions between
fibrils. This may be an additional pathway to the formation
of mature fibrils via association of protofibrils. Conformational
preferences for a certain pathway to become active
may exist, and thus the influence of environmental conditions
such as pH, temperature, and salt must be considered.
Thus, it seems that a single filament may act as a ‘‘lateral
template’’ or scaffold for small protein particles, which
would constitute a neighboring subcord-like feature in the
fiber shown in Fig. 6 h. This filament þ monomers/oligomers
scenario is an alternative pathway to the otherwise
dominating filament þ filament manner of the protein fibril’s
lateral growth, as has been also observed for insulin (57).
AFM data recently reported by Green et al. (91) suggest
that mature fibrils from human amylin are unlikely to be
assembled by the lateral association of protofibrils.
It appears, therefore, that even for a homogeneous HSA
sample undergoing uniform temperature or acidic treatment,
there is still more than one mode of assembly of filaments
(92). Such polymorphism may be caused by differences in
the number of filaments assembled in the mature fibrils;
however, it may also result from the incorporation in different
regions of the sequence of the polypeptide chain with various
types of fibrils. In addition, the differences between the populations
of fibers clearly involve not only the rate of the aggregation
process, but also the different quaternary folds.
Although the different distribution profiles of the fibrillar
features may still be explained in terms of kinetic effects
(e.g., temperature may differently affect the kinetics at various
stages of the assembly, effectively marginalizing certain
sequential processes), high temperatures may also have
a more direct effect on amyloidogenesis, for instance, by
increasing the thermal energy of the interacting molecules
Biophysical Journal 96(6) 2353–2370
161

2368 Juárez et al.
and hence causing the alignment of filaments to become less
accurate. Such unspecific effects may contribute to morphological
differences in any protein’s amyloid samples induced
at high temperature. In this regard, recent reports based on
nuclear magnetic resonance showed that different fibril
morphologies have different underlying secondary structures,
and as such are likely produced by distinct independent
assembly pathways (93,94).
A summary of the various fibril morphologies observed in
this study is shown in Fig. 12 together with a schematic
representation of the assembly process. We propose that at
elevated temperature (except at pH 3.0 in the absence of electrolyte),
HSA forms rapidly globular oligomers that upon
mutual interaction evolve into more elongated structures
(bead-like) that grow to protofibrils either by subsequent
annealing of oligomers and/or protein monomers. Mature
fibrils can be formed by lateral association of protofibrils
or the addition of protein oligomers to the growing fibril,
both at the ends of the fibril and by lateral fusion. Ringlike
structures are present in acidic conditions at elevated
temperature in the presence of electrolyte as an additional
intermediate state formed by association of short bead-like
structures, which disappears when protofibrils are observed
in solution. Thus, we think that they may act as reservoirs
of initially very short protofibrils.
CONCLUSIONS
We observed the formation of protofibrils, curly fibers, and
mature fibrils by the protein HSA under different solution
conditions. We analyzed the fibrillation process and the
conformational changes associated with it by using different
spectroscopic techniques, and confirmed the necessary development
of b-sheet structure upon fibrillation. In addition, the
shapes of the different structural intermediates and final products
in the fibrillation process were observed by TEM, SEM,
and AFM. The obtained fibrils show structural features typical
of classical amyloid fibers, as denoted by XRD, CD, and fluorescence
spectroscopies, and TEM. A model of fibril formation
based on the elongation of protein oligomers through
mutual interactions and subsequent annealing and growth is
FIGURE 12 Mechanisms of fibril formation for HSA.
Biophysical Journal 96(6) 2353–2370
presented. Nevertheless, some differences in the fibrillation
mechanism occur depending on the solution conditions; for
example, ring-shaped structures are observed only as a structural
intermediate under acidic conditions in the presence of
added electrolyte.
SUPPORTING MATERIAL
Ten figures and a table are available at http://www.biophysj.org/biophysj/
supplemental/S0006-3495(09)00322-1.
We thank Dr. Eugenio Vázquez for his assistance with the CD measurements.
This study was supported by the Ministerio de Educación y Ciencia (project
MAT-2007-61604).
REFERENCES
1. Reches, M., and E. Gazit. 2003. Casting metal nanowires within discrete
self-assembled peptide nanotubes. Science. 300:625–627.
2. Rajagopal, K., and J. P. Schneider. 2004. Self-assembling peptides and
proteins for nanotechnological applications. Curr. Opin. Struct. Biol.
14:480–486.
3. Stefani, M., and C. M. Dobson. 2003. Protein aggregation and aggregate
toxicity: new insights into protein folding, misfolding diseases
and biological evolution. J. Mol. Med. 81:678–699.
4. Soto, C., L. Estrada, and J. Castilla. 2006. Amyloids, prions and the
inherent infectious nature of misfolded protein aggregates. Trends
Biochem. Sci. 31:150–155.
5. Khurana, R., C. Ionescu-Zanetti, M. Pope, J. Li, L. Nelson, et al. 2003.
A general mode for amyloid fibril assembly based on morphological
studies using atomic force microscopy. Biophys. J. 85:1135–1144.
6. Chiti, F., M. Stefani, N. Taddei, G. Ramponi, and C. M. Dobson. 2003.
Rationalization of the effects of mutations on peptide and protein aggregation
rates. Nature. 424:805–808.
7. Williams, A. D., E. Portelius, I. Kheterpal, J. T. Guo, K. D. Cook, et al.
2004. Mapping a b amyloid fibril secondary structure using scanning
proline mutagenesis. J. Mol. Biol. 335:833–842.
8. Hortscansky, P., T. Christopeit, V. Schroeckh, and M. Fändrich. 2005.
Thermodynamic analysis of the aggregation propensity of oxidized
Alzheimer’s b-amyloid variants. Protein Sci. 14:2915–2918.
9. Fändrich, M., V. Forge, K. Buder, M. Kittler, C. M. Dobson, et al. 2003.
Myoglobin forms amyloid fibrils by association of unfolded peptide
segments. Proc. Natl. Acad. Sci. USA. 100:15463–15468.
10. Gazit, E. 2002. The ‘‘correctly folded’’ state of proteins: is it a metastable
state? Angew. Chem. Int. Ed. Engl. 41:257–259.
11. Gillmore, J. D., A. J. Stangou, G. A. Tennet, D. R. Booth, J. O’Grady,
et al. 2001. Clinical and biochemical outcome of hepatorenal transplantation
for hereditary systemic amyloidosis associated with apolipoprotein
AI Gly26Arg. Transplantation. 71:986–992.
12. Carulla, N., G. L. Caddy, D. R. May, J. Zurdo, M. Gairi, et al. 2005.
Molecular recycling within amyloid fibrils. Nature. 436:554–558.
13. Pepys, M. B., J. Herbert, W. L. Hutchison, G. A. Tennent, H. J. Lachmann,
et al. 2002. Targeted pharmacological depletion of serum amyloid
P component for treatment of human amyloidosis. Nature. 417:254–259.
14. Janus, C., M. A. Chishti, and D. Westaway. 2000. Transgenic mouse
models of Alzheimer’s disease. Biochim. Biophys. Acta. 1502:63–75.
15. Cohen, F. E., and J. W. Kelly. 2003. Therapeutic approaches to proteinmisfolding
diseases. Nature. 426:905–909.
16. Tanaka, M., P. Chien, N. Narber, R. Cooke, and J. S. Weissman. 2004.
Conformational variations in an infectious protein determine prion
strain differences. Nature. 428:323–328.
162

Fibrillation Pathway of HSA 2369
17. Fändrich, M., and C. M. Dobson. 2002. The behavior of polyamino
acids reveals an inverse side chain effect in amyloid structure formation.
EMBO J. 21:5682–5690.
18. Gorinstein, S., A. Caspi, A. Rosen, I. Goshev, M. Zemser, et al. 2002.
Structure characterization of human serum aproteins in solution and dry
state. J. Pept. Res. 59:71–78.
19. Taboada, P., S. Barbosa, E. Castro, and V. Mosquera. 2006. Amyloid
fibril formation and other aggregate species formed by human serum
albumin association. J. Phys. Chem. B. 110:20733–20736.
20. Pace, C. N., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1995. How to
measure and predict the molar absorption coefficient of a protein.
Protein Sci. 4:2411–2423.
21. Sreerama, N., and R. W. Woody. 2000. Estimation of protein secondary
structure from circular dichroism spectra: comparison of CONTIN,
SELCON, and CDSSTR methods with an expanded reference set.
Anal. Biochem. 287:252–260.
22. Uversky, V. N., and A. L. Fink. 2004. Conformational constraints for
amyloid fibrillation: the importance of being unfolded. Biochim.
Biophys. Acta. 1698:131–153.
23. Calamai, M., F. Chiti, and C. M. Dobson. 2005. Amyloid fibril formation
can proceed from different conformations of a partially unfolded
protein. Biophys. J. 89:4201–4210.
24. Morel, B., S. Casares, and F. A. Conejo-Lara. 2006. Single mutation
induces amyloid aggregation in the a-spectrin SH3 domain: analysis
of the early stages of fibril formation. J. Mol. Biol. 356:453–468.
25. Dockal, M., D. C. Carter, and F. Rüker. 2000. Conformational transitions
of the three recombinant domains of human serum albumin
depending on pH. J. Biol. Chem. 275:3042–3050.
26. Peters, T. Jr., 1996. All About Albumin: Biochemistry, Genetics and
Medical Application. Academic Press, New York.
27. Geisow, M. J., and G. H. Beaven. 1977. Physical and binding properties
of large fragments of human serum albumin. Biochem. J. 165:477–484.
28. Khan, M. Y. 1986. Direct evidence for the involvement of domain III in
the N-F transition of bovine serum albumin. Biochem. J. 236:307–310.
29. Ahmad, B., M. K. A. Khan, S. Haq, and R. H. Khan. 2004. Intermediate
formation at lower urea concentration in ‘B’ isomer of human serum
albumin: a case study using domain specific ligands. Biochem. Biophys.
Res. Commun. 314:166–173.
30. Taboada, P., S. Barbosa, E. Castro, M. Gutiérrez-Pichel, and V. Mosquera.
2007. Effect of solvation on the structure conformation of human
serum albumin in aqueous-alcohol mixed solvents. Chem. Phys.
340:59–68.
31. Farruggia, B., F. Rodriguez, R. Rigatuso, G. Fidelio, and G. Picó. 2001.
The participation of human serum albumin domains in chemical and
thermal unfolding. J. Protein Chem. 20:81–89.
32. Flora, K., J. D. Brennan, G. A. Baker, M. A. Doody, and F. V. Bright.
1998. Unfolding of acrylodan-labeled human serum albumin probed
by steady-state and time resolved fluorescence methods. Biophys. J.
75:1084–1096.
33. Rader, A. J., B. M. Hespenheide, K. A. Kuhn, and M. F. Thorpe. 2002.
Protein unfolding: rigidity lost. Proc. Natl. Acad. Sci. USA. 99:
3540–3545.
34. LeVine, H. 3rd. , 1993. Thioflavine T interaction with synthetic
Alzheimer’s disease b-amyloid peptides: detection of amyloid aggregation
in solution. Protein Sci. 2:404–410.
35. Klunk, W. E., J. W. Pettegrew, and D. J. Abraham. 1989. Quantitative
evaluation of Congo Red binding to amyloid-like proteins with
a b-pleated sheet conformation. J. Histochem. Cytochem. 37:1273–1281.
36. Khurana, R., C. Coleman, C. Ionescu-Zanetti, S. A. Carter, V. Krishna,
et al. 2005. Mechanisms of thioflavin T binding to amyloid fibrils.
J. Struct. Biol. 151:229–238.
37. Sagis, L. M. C., C. Veerman, and E. Van der Linden. 2004. Mesoscopic
properties of semiflexible amyloid fibrils. Langmuir. 20:924–927.
38. Morozova-Roche, L. A., J. A. Jones, W. Noppe, and C. M. Dobson.
1999. Independent nucleation and heterogeneous assembly of structure
during unfolding of equine lysozime. J. Mol. Biol. 289:1055–1073.
39. Jund, P., R. Jullien, and I. Campbell. 2001. Random walks on fractals
and stretched exponential relaxation. Phys. Rev. E. 63:361311–361314.
40. Harper, J. D., and P. T. J. Landsbury. 1997. Models of amyloid seeding
in Alzheimer’s disease and scrapie: mechanistic truths and physiological
consequences of the time-dependent solubility of amyloid proteins.
Annu. Rev. Biochem. 66:385–407.
41. Lomakin, A., D. S. Chung, G. B. Benedek, D. A. Kirschner, and
D. B. Teplow. 1996. On the nucleation and growth of amyloid bprotein
fibrils: detection of nuclei and quantitation of rate constants.
Proc. Natl. Acad. Sci. USA. 93:1125–1129.
42. Lomakin, A., D. B. Teplow, D. A. Kirschner, and G. B. Benedek. 1997.
Kinetic theory of fibrillogenesis of amyloid b-protein. Proc. Natl. Acad.
Sci. USA. 94:7942–7947.
43. Ferrone, F. 1999. Analysis of protein aggregation kinetics. Methods
Enzymol. 309:256–274.
44. Susi, H., and D. M. Byler. 1983. Protein structure by Fourier transform
infrared spectroscopy: second derivative spectra. Biochem. Biophys.
Res. Commun. 115:391–397.
45. Lin, S. -Y., Y. -S. Wei, M. -J. Li, and S. -L. Wang. 2004. Effect of ethanol
or/and captopril on the secondary structure of human serum albumin
before and after protein binding. Eur. J. Pharm. Biopharm. 57:457–464.
46. Jackson, M., and H. H. Mantsch. 1995. The use and misuse of
FTIR spectroscopy in the determination of protein structure. Crit.
Rev. Biochem. Mol. Biol. 30:95–120.
47. Casal, H. L., U. Kohler, and H. H. Mantsch. 1988. Structural and
conformational changes of b-lactoglobulin B: an infrared spectroscopy
study of the effect of pH and temperature. Biochim. Biophys. Acta.
957:11–20.
48. Fabian, H., L. -P. Choo, G. I. Szendrei, M. Jackson, W. C. Halliday,
et al. 1993. Infrared spectroscopic characterization of Alzheimer
plaques. Appl. Spectrosc. 47:1513–1518.
49. Lin, S. -Y., Y. -S. Wei, M. -J. Li, and S. -L. Wang. 2004. Effect of
ethanol or/and captopril on the secondary structure of human serum
albumin before and after protein binding. Spectrochim. Acta [A].
60:3107–3111.
50. Uversky, V. N., N. V. Narizhneva, T. V. Ivanova, and A. Y. Tomashevski.
1997. Rigidity of human a-fetoprotein tertiary structure is under
ligand control. Biochemistry. 494:13638–13645.
51. Muzammil, S., Y. Kumar, and S. Tayyab. 1999. Molten globule-like
state of human serum albumin at low pH. FEBS Lett. 266:26–32.
52. Qiu, W., L. Zhang, O. Okobiah, Y. Yang, L. Wang, et al. 2006. Ultrafast
solvation dynamics of human serum albumin: correlations with conformational
transitions and site-selected recognition. J. Phys. Chem. B.
110:10540–10549.
53. Cowgill, R. W. 1968. Fluorescence and protein structure. XIV. Tyrosine
fluorescence in helical muscle proteins. Biochim. Biophys. Acta.
161:431–438.
54. Shahi, P., R. Sharma, S. Sanger, I. Kumar, and R. S. Jolly. 2007. Formation
of amyloid fibrils via longitudinal growth of oligomers. Biochemistry.
46:7365–7373.
55. Burgio, M. R., P. M. Bwennet, and J. F. Koretz. 2001. Heat-induced
quaternary transitions in hetero- and homo-polymers of a-crystallin.
Mol. Vis. 7:228–233.
56. Meehan, S., Y. Berry, B. Luisa, C. M. Dobson, J. A. Carver, et al. 2004.
Amyloid fibril formation by lens crystallin proteins and its implications
for cataract formation. J. Biol. Chem. 279:3413–3419.
57. Jansen, R., W. Dzwolak, and R. Winter. 2005. Amyloidogenic selfassembly
of insulin aggregates probed by high resolution atomic force
microscopy. Biophys. J. 88:1344–1353.
58. Radford, S. E., W. S. Gosal, and G. W. Platt. 2005. Towards an understanding
of the structural molecular mechanism of b2-microglobulin
amyloid formation in vitro. Biochim. Biophys. Acta. 1753:51–63.
59. Kodali, R., and R. Wetzel. 2007. Polymosphism in the intermediates
and products of amyloid assembly. Curr. Opin. Struct. Biol. 17:48–57.
60. Sunde, M., and C. Blake. 1997. The structure of amyloid fibrils by electron
microscopy and X-ray diffraction. Adv. Protein Chem. 50:123–159.
Biophysical Journal 96(6) 2353–2370
163

2370 Juárez et al.
61. Bouchard, M., J. Zurdo, E. J. Nettleton, C. M. Dobson, and C. V. Robinson.
2000. Formation of insulin amyloid fibrils followed by FTIR
simultaneously with CD and electron microscopy. Protein Sci.
9:1960–1967.
62. Vetri, V., F. Librizzi, M. Leone, and V. Militello. 2007. Thermal aggregation
of bovine serum albumin at different pH: comparison with
human serum albumin. Eur. Biophys. J. 36:717–725.
63. Bader, R., R. Bamford, J. Zurdo, B. F. Luisi, and C. M. Dobson. 2006.
Probing the mechanism of amyloidogenesis through a tandem repeat of
the PI3–SH3 domain suggests a generis model for protein aggregation
and fibril formation. J. Mol. Biol. 356:189–208.
64. Shaw, A. K., and S. K. Pal. 2008. Spectroscopic studies on the effect
of temperature on pH-induced folded states of human serum albumin.
J. Photochem. Photobiol. B. 90:69–77.
65. Ahmad, B., S. Parveen, and R. H. Khan. 2006. Effect of albumin
conformation on the binding of ciprofloxacin to human serum albumin:
a novel approach directly assigning binding site. Biomacromolecules.
7:1350–1356.
66. Eftink, M. R., and C. A. Ghiron. 1976. Exposure of tryptophyl residues
in proteins. Quantitative determination by fluorescence quenching
studies. Biochemistry. 15:672–679.
67. Sibley, S. P., K. Sosinsky, L. E. Gulian, E. J. Gibbs, and R. F. Pasternack.
2008. Probing the mechanism of insulin aggregation with added
metalloporphyrins. Biochemistry. 47:2858–2865.
68. Zheng, J., H. Jang, B. Ma, and R. Nussinov. 2008. Annular structures as
intermediates in fibril formation of Alzheimer Ab17–42. J. Phys. Chem.
B. 112:6856–6865.
69. Moore, R. E., S. F. Hayes, E. R. Fischer, and S. A. Priola. 2007.
Amyloid formation via supramolecular peptide assemblies. Biochemistry.
46:7079–7087.
70. Malisauskas, M., V. Zamotin, J. Jass, W. Noppe, C. M. Dobson, et al.
2003. Amyloid protofilaments from the calcium-binding protein equine
lysozyme: formation of ring and linear structures depends on pH and
metal ion concentration. J. Mol. Biol. 330:879–890.
71. Lashuel, H. A., D. Hartley, B. M. Petre, T. Walz, and P. T. Lansbury Jr.
2002. Neurodegenerative disease: amyloid pores from pathogenic mutations.
Nature. 418:291.
72. Jarrett, J. T., and P. T. Lansbury Jr. 1992. Amyloid fibril formation
requires a chemically discriminating nucleation event—studies of an
amyloidogenic sequence from the bacterial protein OSMB. Biochemistry.
31:12345–12352.
73. Lefebvre, J., D. Renard, and A. C. Sánchez-Gimeno. 1998. Structure
and rheology of heat-set gels of globular proteins. I. Bovine serum
albumin gels in isoelastic conditions. Rheol. Acta. 37:345–357.
74. Holm, N. K., S. K. Jespersen, L. V. Thomassen, T. Y. Wolff, P. Sehgal,
et al. 2007. Aggregation and fibrillation of bovine serum albumin. Biochim.
Biophys. Acta. 1774:1128–1138.
75. Chiti, F., P. Webster, N. Taddei, A. Clark, M. Stefani, et al. 1999.
Designing conditions for in vitro, formation of amyloid protofilaments
and fibrils. Proc. Natl. Acad. Sci. USA. 96:3590–3594.
76. Krishnan, R., and S. L. Lindquist. 2005. Structural insights into a yeast
prion illuminate nucleation and strain diversity. Nature. 435:765–772.
77. Moraitakis, G., and J. M. Goodfellow. 2003. Simulations of human lysozime:
probing the conformations triggering amyloidosis. Biophys. J.
84:2149–2158.
Biophysical Journal 96(6) 2353–2370
78. Modler, A. J., K. Gast, G. Lutsch, and G. Damaschun. 2003. Assembly
of amyloid protofibrils via critical oligomers. A novel pathway of
amyloid formation. J. Mol. Biol. 325:135–148.
79. Serio, T. R., A. G. Cashikar, A. S. Kowal, G. J. Sawicki, J. J. Moleshi,
et al. 2000. Nucleated conformational conversion and the replication of
conformational information by a prion determinant. Science. 289:
1317–1321.
80. Jiang, Y., H. Li, L. Zhu, J. M. Zhou, and S. Perrett. 2004. Amyloid
nucleation and hierarchical assembly of Ure2p fibrils. Role of asparagines/glutamine
repeat and nonrepeat regions of the prion domains.
J. Biol. Chem. 279:3361–3369.
81. Makin, O. S., and L. C. Serpell. 2005. X-ray diffraction studies of
amyloid structure. Methods Mol. Biol. 299:67–80.
82. Sambashivan, S., M. R. Liu, M. R. Sawaya, M. Gingery, and D. Eisenberg.
2005. Amyloid-like fibrils of ribonuclease A with threedimensional
domain-swapped and native-like structures. Nature.
437:266–269.
83. Ranson, N., T. Stromer, L. Bousset, R. Melki, and R. C. Serpell. 2006.
Insights into the architecture of the Ure2p yeats protein assemblies from
helical twisted fibrils. Protein Sci. 15:2481–2487.
84. Hatters, D. M., C. A. MacRails, R. Daniels, W. S. Gosal, N. H. Thomson,
et al. 2003. The circularization of amyloid fibrils formed by apolipoprotein
C–II. Biophys. J. 85:3979–3990.
85. Lashuel, H. A., and P. T. Lansbury Jr. 2006. Are amyloid diseases
caused by protein aggregates that mimic bacterial pore-forming toxins?
Q. Rev. Biophys. 39:167–201.
86. Meehan, S., T. P. J. Knowles, A. J. Baldwin, J. F. Smith, A. M. Squires,
et al. 2007. Characterization of amyloid fibril formation by small heatshock
chaperone proteins human aA-, aB- and R120G aB-crystallins.
J. Mol. Biol. 372:470–484.
87. Kad, N. M., S. L. Myers, D. P. Smith, S. E. Radford, and N. H. Thomson.
2003. Hierarchical assembly of b2-microglobulin amyloid in vitro
revealed by atomic force microscopy. J. Mol. Biol. 330:785–797.
88. Khurana, R., J. L. Jimenez, E. J. Nettleton, M. Bouchard, C. V. Robinson,
et al. 2002. The protofilament structure of insulin amyloid fibrils.
Proc. Natl. Acad. Sci. USA. 99:9196–9201.
89. Otzen, D. E., and M. Oliveberg. 2004. Transient formation of nanocrytalline
structures during fibrillation of an Alzheimer-like peptide.
Protein Sci. 13:1417–1421.
90. Grudzielanek, S., V. Smirnovas, and R. Winter. 2006. Solvation-assisted
pressure tuning of insulin fibrillation: from novel aggregation pathways
to biotechnological applications. J. Mol. Biol. 356:497–506.
91. Green, J. D., C. Goldsbury, J. Kistler, G. K. Cooper, and U. Aebi. 2004.
Human amylin oligomer growth and fibril elongation define two distinct
phases in amyloid formation. J. Biol. Chem. 279:12206–12212.
92. Chamberlain, A. K., C. E. MacPhee, J. Zurdo, L. A. Morozova-Roche,
H. A. O. Hill, et al. 2000. Ultrastructural organization of amyloid fibrils
by atomic force microscopy. Biophys. J. 79:3282–3293.
93. Goldsbury, C. S., S. Wirtz, S. A. Müller, S. Sunderji, P. Wicki, et al.
2000. Studies on the in vitro assembly of Aba-40: implications for
the search for Ab fibril formation inhibitors. J. Struct. Biol. 130:
217–231.
94. Petkova, A. T., R. D. Leapman, Z. Guo, W. M. Yau, M. P. Mattson,
et al. 2005. Self-propagating, molecular-level polymorphism in
Alzheimer’s b-amyloid fibrils. Science. 307:262–265.
164

Influence of Electrostatic Interactions on the Fibrillation Process of Human Serum Albumin
Introduction
Josué Juárez, Sonia Goy López, Adriana Cambón, Pablo Taboada,* and Víctor Mosquera
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada, Facultad de
Física, UniVersidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain
ReceiVed: March 12, 2009; ReVised Manuscript ReceiVed: May 7, 2009
The fibrillation propensity of the multidomain protein human serum albumin (HSA) has been analyzed under
physiological and acidic conditions at room and elevated temperatures with varying ionic strengths by different
spectroscopic techniques. The kinetics of fibril formation under the different solution conditions and the
structures of resulting fibrillar aggregates were also determined. In this way, we have observed that fibril
formation is largely affected by electrostatic shielding: at physiological pH, fibrillation is progressively more
efficient and faster in the presence of up to 50 mM NaCl; meanwhile, at larger salt concentrations, excessive
shielding and further enhancement of the solution hydrophobicity might involve a change in the energy
landscape of the aggregation process, which makes the fibrillation process difficult. In contrast, under acidic
conditions, a continuous progressive enhancement of HSA fibrillation is observed as the electrolyte concentration
in solution increases. Both the distinct ionization and initial structural states of the protein before incubation
may be the origin of this behavior. CD, FT-IR, and tryptophan fluorescence spectra seem to confirm this
picture by monitoring the structural changes in both protein tertiary and secondary structures along the
fibrillation process. On the other hand, the fibrillation of HSA does not show a lag phase except at pH 3.0 in
the absence of added salt. Finally, differences in the structure of the intermediates and resulting fibrils under
the different conditions are also elucidated by TEM and FT-IR.
Protein aggregation and, particularly, amyloid formation have
received considerable interest in the fields of protein research
and clinical medicine, since growing evidence has accumulated
that these processes are likely to be key issues in the etiology
of various medical disorders such as Alzheimer’s, Parkinson’s,
Huntington’s, and Creutzfeldt-Jakob diseases, type II diabetes,
and cystic fibrosis among others. In fact, about 20 different
known syndromes are associated with the formation of amyloid
fibrils. 1-6 In addition, a number of nondisease associated proteins
have also been found to be able to form ordered cytotoxic
aggregates and amyloid fibrils in Vitro. 7 On the other hand, the
exceptional physical characteristics of the amyloidal protein
state, such as its stability, mechanical strength, and resistance
to degradation, imply that this type of structure possesses a range
of potential technological applications in biotechnology and
materials science. 8,9
A consensus has emerged that the different processes of in
ViVo and in Vitro aggregation might be linked by the implication
of destabilized protein molecules that undergo non-native
backbone-dominated self-assembly as a competing pathway to
native functional folding. 10 However, both folding and protein
aggregation are thought to underlie some common principles
which aim at minimizing the Gibbs energy of destabilized
protein states, such as the saturation of dangling hydrogen
bonds, 11 the reduction of the solvent-accessible surface area, or
the burial of hydrophobic residues. 12 In this way, globular
proteins generally need to be destabilized (e.g., by mutation, 13,14
heat, 15,16 high pressure, 17,18 low pH, 19,20 or organic denaturant 7,15 )
to aggregate rapidly, with fibril formation proceeding from
* Author to whom correspondence should be addressed. E-mail:
pablo.taboada@usc.es. Telephone: 0034981563100, ext. 14042. Fax:
0034981520676.
J. Phys. Chem. B 2009, 113, 10521–10529 10521
extensively or partially unfolded states 3,21 or, in some cases,
from native-like states in which unfolding may initially be
limited and confined to local regions of the protein. 22,23
The physiological importance of human serum albumin as a
carrier protein and blood pressure regulator and its propensity
to easily aggregate in Vitro have become a good model for
protein aggregation studies. As the phenomenon of protein
aggregation appears to reflect certain generic “polymeric”
features of proteins, 24 the studies of the mechanisms of protein
aggregation on model systems are extremely useful for a better
understanding of the molecular mechanisms of disease-associated
amyloidogenesis. Since in living organisms the native
environment of proteins is a complex composition of water,
cosolvents, and cosolutes which affect the stability of the native
protein fold, 25 experimental approaches to mimic various cellular
environments in Vitro to allow investigations on protein folding
and aggregation often utilize the addition of cosolvents and
cosolutes. Thereby, differences in the properties of the solvent
might induce solvent-adapted structural “responses” of the
aggregating protein. In particular, the importance of electrostatic
interactions in the formation of amyloid fibrils 20,26,27 and the
relevance of the total net charge on the protein to the fibril
formation propensity have been pointed out. 26,27
To put more light on this issue, in the present work we present
a systematic study on the relationship between serum albumin
amyloid-like fibrillation kinetics, fibrillation pathways, and the
resulting aggregated protein structures under different conditions
where the amyloid fibrils of this model protein were found by
changing solution pH and solution ionic strength. 28,29 In this
manner, a modulation of the electrostatic interactions between
protein molecules and aggregates results in different balances
of intermolecular forces between protein molecules, which
involve changes in both protein aggregation and their kinetics.
In this way, we have found large differences in the fibrillation
10.1021/jp902224d CCC: $40.75 © 2009 American Chemical Society
Published on Web 07/02/2009
165

10522 J. Phys. Chem. B, Vol. 113, No. 30, 2009 Juárez et al.
kinetics of HSA and, as a result, different structural intermediates
and fibrillation pathways. As well as that, differences in
the resulting amyloid-like fibrils under the different solution
conditions were also observed. It seems, then, that both pH and
ionic strength play a key role in regulating the several steps
which regulate HSA fibrillation.
Experimental Section
Materials. Human serum albumin (70024-90-7), Congo Red
(CR), and Thioflavin T (ThT) were obtained from Sigma
Chemical Co and used as received. All other chemicals were
of the highest purity available.
Preparation of HSA Solutions. The buffer solutions used
were glycine + HCl for pH 3.0 and sodium monophosphatesodium
diphosphate for pH 7.4, respectively. The buffers were
prepared in different NaCl concentrations (0, 20, 50, 100, 150,
and 250 mM NaCl). The HSA was dissolved in each buffer
solution to a final concentration of typically 20 mg/mL, if not
otherwise stated. Protein concentration was determined spectrophotometrically
using a molar absorption coefficient of 35 219
M-1 cm-1 at 280 nm. 30 Before incubation, the solution was
filtered through a 0.2 µm filter into sterile test tubes. Samples
were incubated at a specified temperature in a refluxed reactor.
Samples were taken out at intervals and stored on ice before
adding CR or ThT.
Seeding Solutions. To test whether seeding with preformed
aggregates increases the rate of HSA aggregation under the
different conditions where fibrils are formed, a protein solution
was incubated for 24-48 h and an aliquot that corresponded to
10% (w/w) of the total protein concentration was then added
to a fresh protein solution.
Thioflavin T Spectroscopy. 50 µL aliquots of protein
solution were withdrawn at different times and diluted 100 times
with 4 mL of ThT solution in corresponding buffer (final ThT
concentration 20 µM). Samples were continuously stirred during
measurements. Fluorescence was measured in a Cary Eclipse
fluorescence spectrophotometer equipped with a temperature
control device and a multicell sample holder (Varian Instruments
Inc.). Excitation and emission wavelengths were 450 and 482
nm, respectively. All intensities were background-corrected for
the ThT-fluorescence in the respective buffer without the protein.
Congo Red Binding. Changes in the absorbance of Congo
Red dye produced by binding to HSA were measured in a
UV-vis spectrophotometer DU Series 640 (Beckman Instruments)
operating from 190 to 1100 nm. All measurements were
made in the wavelength range 200-600 nm in matched quartz
cuvettes. Protein solutions were diluted 20-200-fold into a
buffer solution with 5 µM Congo Red (Acros Organics, Geel,
Belgium). Spectra in the presence of the dye were compared
with that of the buffer containing Congo Red in the absence of
protein, and also with that containing protein without dye.
Circular Dichroism (CD). Far-UV circular dichroism (CD)
spectra were obtained using a JASCO-715 automatic recording
spectropolarimeter with a JASCO PTC-343 Peltier-type thermostatted
cell holder. Quartz cuvettes with 0.2 cm path length
were used. CD spectra were obtained from aliquots withdrawn
from the aggregation mixtures at the indicated conditions and
incubation times, and recorded between 195 and 250 nm at 25
°C. The mean residue ellipticity θ (deg cm2 dmol-1 ) was
calculated from the formula θ ) (θobs/10)(MRM/lc), where θobs
is the observed ellipticity in deg, MRM is the mean residue
molecular mass, l is the optical path-length (in cm), and c is
the protein concentration (in g mL-1 ). In order to calculate the
composition of the secondary structure of the protein, SEL-
CON3, CONTIN, and DSST programs were used to analyze
far-UV CD spectra. Final results were assumed when data
generated from all programs show convergence. 31
Fourier Transform Infrared Spectroscopy (FT-IR). FT-
IR spectra of HSA in aqueous solutions were determined by
using a FT-IR spectrometer (model IFS-66v from Bruker)
equipped with a horizontal ZnS ATR accessory. The spectra
were obtained at a resolution of 2 cm -1 , and generally 200 scans
were accumulated to get a reasonable signal-to-noise ratio.
Solvent spectra were also examined in the same accessory and
at the same instrument conditions. Each different sample
spectrum was obtained by digitally subtracting the solvent
spectrum from the corresponding sample spectrum. Each sample
solution was repeated three times to ensure reproducibility and
was averaged to produce a single spectrum.
Protein Fluorescence. To examine the conformational variations
around the tryptophan residue of HSA, fluorescence
emission spectra of HSA samples were excited at 295 nm, which
provides no excitation of tyrosine residues, and therefore, neither
emission nor energy transfer to the lone side chain takes place.
Slit widths were typically 5 nm. The spectrophotometer used
was a Cary Eclipse from Varian Instruments Inc.
Transmission Electron Microscopy (TEM). Suspensions of
HSA were applied to carbon-coated copper grids, blotted,
washed, negatively stained with 2% (w/v) of phosphotungstic
acid, air-dried, and then examined with a Phillips CM-12
transmission electron microscope operating at an accelerating
voltage of 120 kV. Samples were diluted between 20 and 200fold
where prior deposition on the grids was needed.
X-ray Diffraction. X-ray diffraction experiments were carried
out using a Siemens D5005 rotating anode X-ray generator.
Twin Göbel mirrors were used to produce a well-collimated
beam of Cu KR radiation (λ ) 1.5418 A). Samples were put
into capillaries with diameters of 0.5 mm. X-rays diffraction
patterns were recorded with an AXS F.Nr. J2-394 imaging plate
detector.
Results and Discussion
It has been previously reported that amyloid fibrils can also
be formed in Vitro from R-globular proteins such as myoglobin,
ovoalbumin,bovine(BSA),andhuman(HSA)serumalbumins, 4,32,33
for example. Similar to these proteins, native HSA also lacks
any properties suggesting a predisposition to form amyloid
fibrils, since most of its sequence (>60%) is arranged in an
R-helix structure, with the subsequent tightening of its structure
through intramolecular interactions such as hydrogen bonds. In
this way, serum albumin aggregation is only promoted under
conditions favoring partly destabilized monomers and dimers
such as low pH, high temperatures, or the presence of chemical
denaturants. 34 In a recent report, 28 we have shown that partially
destabilized HSA molecules form amyloid-like fibrils and other
types of aggregates under different solution conditions such as
increasing the solution temperature and changing the solution
pH. 28,35,36 Thus, HSA was subjected to conditions previously
found to be effective for protein aggregation, in particular those
inducing amyloid-like fibril formation but modifying the solution
ionic strength in order to analyze its effects on the amyloidlike
assembly pathway of this protein. Thus, we prepared HSA
solutions at a concentration of 20 mg/mL in 0.01 M sodium
phosphate buffer (pH 7.4) or 0.01 M glycine-HCl buffer (pH
3.0) in the presence of 0, 20, 50, 100, 150, and 250 mM NaCl
at 25 or 65 °C followed by subsequent incubation for up to 15
days. HSA is in its native form (N) at pH 7.4, which is composed
mostly of R-helix secondary structure. In contrast, HSA adopts
166

Fibrillation Process of Human Serum Albumin J. Phys. Chem. B, Vol. 113, No. 30, 2009 10523
Figure 1. Time evolution of ThT fluorescence in HSA solutions
incubated at 65 °C at (a) pH 7.4 and (b) pH 3.0 in the presence of (9)
0, (O) 20, (2) 50, (3) 100, ([) 150, and (+) 250 mM NaCl.
an extended less-ordered configuration (E-state) below pH 3.5
with lesser helical content. 37-39 On the other hand, 65 °C was
chosen as the incubation temperature, provided that HSA
temperature-induced denaturation takes place through a twostate
transition with a first melting temperature, Tm, of∼56 °C
and a second Tm of ∼62 °C 35,40,41 as a consequence of the
sequential unfolding of the different domains of the protein, in
particular IIA and IIIA subdomains: at 50 °C, a reversible
separation of site I and II occurs; below 70 °C, the irreversible
unfolding of site II is present, while increasing the temperature
over 70 °C or higher results in the irreversible unfolding
of site I. 42 Moreover, as the pH becomes more acidic, Tm
becomes lower. 35
Amyloid Formation Kinetics. When HSA solutions (20 mg/
mL in the presence of 0, 20, 50, 100, 150, or 250 mM NaCl)
were incubated at room temperature up to 300 h, the ThT
fluorescence emission intensity was negligible at pH 7.4 whereas
a slight increase was observed at pH 3.0 as a consequence of a
little gain in -sheet structure due to the formation of some
oligomeric aggregates (data not shown). This suggests that HSA
was not capable of forming amyloid fibrils or other types of
aggregates with significant -sheet structure under these conditions.
In contrast, a time-dependent increase in ThT fluorescence
is observed when the different HSA solutions were incubated
at 65 °C (see Figure 1), in accordance with fibril formation.
Congo Red (CR) absorption was also used to corroborate the
formation of these fibrils in HSA solutions, which display a
red-shift of the differential absorption maximum from 495 to
ca. 530 nm at 65 °C (see Figure S1 as an example).
In general, the kinetics of HSA fibrillation under the present
solution conditions at 65 °C involved the continuous rising of
TABLE 1: Kinetic Parameters of the Self-Assembly Process
of HSA Solutions
pH NaCl (mM) ∆F ksp (h-1 ) n
7.4 0 83 0.019 0.56
20 82 0.040 0.70
50 83 0.084 0.92
100 57 0.068 0.80
150 52 0.056 0.77
250 46 0.034 0.70
3.0 0 6/22 0.144/0.005 1.25/3.60
20 44 0.008 1.25
50 57 0.010 0.96
100 73 0.012 0.95
150 77 0.024 0.93
250 56 0.233 1.10
ThT fluorescence during the early periods of incubation until a
quasi-plateau region was attained in the time scale analyzed,
as occurred for different proteins such as BSA, 32 acylphosphatase,
7 or ovoalbumin, 33 and it exhibited no discernible lag
phase. This absence may be related to the fact that the initial
protein aggregation is a downhill process, which does not require
a highly organized and stable nucleus. This was confirmed by
the absence of any remarkable effect on the fluorescence curves
when protein seeds are added to protein solutions followed by
subsequent incubation (see Figure S2 in the Supporting Information).
It has been suggested that large multidomain proteins
such as BSA are able to form propagation-competent nucleuslike
structures (oligomeric structures), since there is no energy
barrier to impede aggregate growth. 32 As has been discussed in
detail previously before, 32,36 HSA forms these oligomers upon
very short incubation times, which usually occurs by a mechanism
of classical coagulation or downhill polymerization. 44
Nevertheless, a different behavior was found at acidic pH in
the absence of added electrolyte: After a small increase in ThT
fluorescence at very short incubation times, there exists an
almost plateau region (between 24 and 100 h approximately),
from which the ThT fluorescence starts to increase again (see
Figure 1b). This plateau region was identified as a lag phase,
as confirmed by a fibril seeding growth process (see Figure S3
in the Supporting Information). This might well indicate that
the formation of oligomeric structures might need more time
to be developed and/or persist for longer times due to their
enhanced solubility under acidic conditions in the absence of
added salt, so oligomers need to reach a certain number/size to
change the energy landscape of the system and promote further
aggregation.
Figure 1 also shows that amyloid formation was favored and
took place progressively quicker as the electrolyte concentration
increased from 0 to 50 mM NaCl at pH 7.4 and at the whole
concentration range studied at pH 3.0, as denoted by the
increment in ThT fluorescence intensity and the subsequent
presence of a plateau region at shorter incubation times. This
points out that the increasing hydrophobicity originated from
the screening of repulsive electrostatic interactions between
protein molecules is capable of stabilizing the -sheets. 36,45-48
In contrast, a progressive decrease in ThT fluorescence intensity
is observed at electrolyte concentrations larger than 50 mM at
physiological pH, with the plateau located at shorter incubation
times. This decrease can be related to the formation of bundles
of shorter fibrils and to the presence of amorphous aggregates
(with larger R-helix and unordered conformation contents, as
will be shown below), and is a consequence of an excessive
shielding of intermolecular electrostatic forces, which is accompanied
by an additional enhancement of the solution
167

10524 J. Phys. Chem. B, Vol. 113, No. 30, 2009 Juárez et al.
Figure 2. Far-UV spectra of HSA solutions at 65 °C and pH 7.4 in the presence of (a) 0, (b) 50, (c) 100, and (d) 150 mM NaCl at (1) 0 h, (2)
12 h, (3) 24 h, (4) 48, (5) 60 h, and (6) 72 h of incubation.
hydrophobicity through (a) a side-to-side adsorption between
protein molecules and fibrils; (b) a reduction of the ThT
fluorescence by blocking ThT binding sites on the fibrils due
to a reduction in exposed surface area; 49 and (c) a lower fraction
of formed fibrils due to an unsuitable energy balance to establish
effective intermolecular interactions; that is, random aggregation
is promoted, so there exists a change in the energy landscape
of the protein fibrillation process. 50
Differences in the trends observed between physiological and
acidic conditions in the presence of electrolyte might arise from
the fact that ordered aggregation of globular proteins requires
partial unfolding of the native state, 21 which enables this
precursor state to be populated and to expose aggregationcompetent
regions that are usually protected against intermolecular
interactions in the native protein. However, these regions
can be different and display different propensities to solvent
exposition depending on solution conditions. Thus, although
amyloid formation can occur, rates and extents of fibril
formation, fibrillation pathways, and the structures of the
resulting fibrils can be very diverse. 51,52 In this way, it seems
that both the distinct ionization state of the protein and the
different initial structural state at acidic conditions (expanded-
E-state) may be the origin of the observed differences, which
change the energy landscape of the aggregation process.
The time-dependence of the ThT fluorescence curves was
fitted by nonlinear square curve-fitting to a stretched exponential
function F ) F ∞ + ∆F exp(-[kspt] n ) in order to derive
information on the amyloid kinetics formation. F, F ∞ , and ∆F
are the observed fluorescence intensity at time t, the final
fluorescence intensity, and the fluorescence amplitude, respectively,
and ksp is the rate of spontaneous fibril formation. Values
of ksp, n, and ∆F are shown in Table 1. Values of n lower than
1 indicated that the kinetics can be approximated to several
exponential functions indicative of the existence of multiple
events in the amyloid formation. The increasing ksp values as
the electrolyte concentration goes up to 100 mM at physiological
pH and at the whole concentration range under acidic conditions
corroborate the more efficient aggregation due to electrostatic
screening in the presence of added salt. However, under
physiological conditions, progressively lower ksp and n values
are derived at larger ionic strengths (see Table 1), maybe as a
predominance of fibril bundling and formation of amorphous
aggregates over that of new fibrils, as commented before. This
behavior is in contrast to that observed for other albumin
proteins such as ovoalbumin and BSA, for which their fibrillation
mechanisms and, thus, their aggregation rates were found
not to be so strongly influenced by ionic strength if compared
to that of HSA. 33 In particular, for ovolabumin, it was found
that a continuous slight decrease in the rate of fibrillation takes
place as the electrolyte concentration occurs, whereas for BSA
the trend is similar to that of HSA, although less marked.
Possibly, the subtle differences in the amino acid sequence
between the proteins and in their structural conformation may
account for these differences.
On the other hand, it is interesting to note that, after 3 days
of incubation, most HSA solutions transformed into a gel-like
state at elevated ionic strengths (between 100 and 250 mM). It
is known that salt concentrations have an important role, not
only favoring conformational transitions and intramolecular
interactions in the protein but also enhancing protein-protein
interactions leading to gelification. 48-50 In our case, these gels
can be a consequence of a “correct” balance between electrostatic
shielding and hydrophobic interactions which enables
fibrils to interact in suitable relative orientations, as has been
previously demonstrated for other proteins. 50 Nevertheless, these
effects will be discussed in a forthcoming publication.
168

Fibrillation Process of Human Serum Albumin J. Phys. Chem. B, Vol. 113, No. 30, 2009 10525
Figure 3. Far-UV spectra of HSA solutions at 65 °C and pH 3.0 in the presence of (a) 0, (b) 50, and (c) 150 mM NaCl at (1) 0 h, (2) 24 h, (3)
50 h, (4) 100 h, (5) 150 h, and (6) 250 h of incubation.
TABLE 2: Secondary Structure Variations (in %) on HSA
Samples upon Incubation at 65° C under Different Solution
pH Values and Electrolyte Concentrations
pH NaCl (mM) ∆R-helix ∆-sheet ∆turn ∆ unordered
7.4 0 -35 16 8 11
20 -37 18 9 12
50 -42 21 10 11
100 -39 16 9 14
150 -37 15 8 16
250 -33 11 7 19
3.0 0 -20 9 3 8
20 -25 12 4 9
50 -28 14 4 10
100 -32 17 5 10
150 -36 19 6 11
250 -35 20 5 10
Structural Changes. To shed additional light on the differences
of the amyloid-like formation of HSA in the presence of
added salt at the different pH values, we recorded CD, FT-IR,
and tryptophan (Trp) fluorescence spectra. The far-UV CD
spectra of HSA solutions incubated at room temperature in the
absence and the presence of electrolyte showed two minima at
208 and 222 nm, characteristic of a helical structure. At acidic
pH, HSA molecules also preserve an important part of their
R-helix structure upon incubation, as denoted by the presence
of the minima at 208 and 222 nm. However, a slight decrease
in ellipticity, [θ], is observed, which is compatible with a little
decrease of R-helices due to the appearance of small amorphous
aggregates in solution (not shown).
When HSA solutions were incubated at 65 °C, far-UV CD
spectra showed a dramatic change in ellipticity throughout
incubation. Figures 2 and 3 show CD spectra for HSA solutions
at 65 °C in the presence of several NaCl concentrations at both
pH 7.4 and 3.0, respectively. Under these conditions, the
minimum at 222 nm progressively disappears while the negative
band intensity at 208 nm is also reduced. All this indicates that
high-temperature conditions spawn intermediates, which are
clearly less helical than the starting conformations (a decrease
in R-helix content is observed). Thus, these changes obtained
in CD spectra suggest the increments of -sheet and loop
structures at longer incubation times (∼72 h and ∼150 h for
pH 7.4 and 3.0, respectively), which resemble those typical of
proteins with important proportions of -sheet structure characterized
by a minimum at ca. 215-220 nm and small [θ] values
as a consequence of the presence of -rich aggregates in
solution, as can be observed, for example, in Figure 2b.
Nevertheless, it is worth mentioning that the increasing number
and size of large scattering objects such as fibrils in solution as
the incubation proceeds involves the strong decrease in ellipticity,
which makes it a little less clear the ellipticity minimum
around 215-220 nm corresponding to -sheet structures.
Changes in protein structure composition derived at the end
of incubation at 65 °C were obtained from CD analysis 31 and
are shown in Table 2. Data in this table are displayed as the
difference (expressed in %) between the beginning and the end
of the incubation process of protein structure composition (∆Rhelix,
∆-sheet, ∆turn, and ∆random coil conformation, respectively).
The negative signs mean a decrease in the corresponding
structure. At pH 7.4 and room temperature, the initial
R-helix content is ∼59%, the -sheet conformation is ∼5%,
the turn content is ∼13%, and the remaining random coil content
is ∼23%, in agreement with previous reports. 47 When HSA
solutions were incubated at 65 °C, these showed important
differences in the secondary structure compositions (see Table
2). The largest changes were observed in R-helix and -sheet
169

10526 J. Phys. Chem. B, Vol. 113, No. 30, 2009 Juárez et al.
Figure 4. Second derivative of FT-IR spectra at 65 °C and (a) pH 7.4 and (b) pH 3.0 in the presence of (1) 0, (2) 20, (3) 50, (4) 100, (5) 150, and
(6) 250 mM NaCl after incubation.
Figure 5. Time evolution of Trp fluorescence of HSA solutions upon
incubation at (a) pH 7.4 and (b) pH 3.0 in the presence of (9) 0,(O)
20, (∆) 50, (1) 100, and (]) 150 mM, and (b) at pH 3.0 and 25 °C in
the presence of 50 mM NaCl.
contents, with the former mainly decreased to be converted into
the -sheet structure. Moreover, coil and turn conformations
also change throughout the incubation process, increasing
slightly their significance.
On the other hand, increases in the -sheet content become
more important as the electrolyte concentration increases, but
only up to 50 mM NaCl at physiological conditions and in the
whole concentration range at acidic pH, for which these
variations were lighter. In this regard, fully mature amyloid
fibrils could not be formed at low electrolyte concentrations at
acidic pH, provided that effective interactions between -strands
are not allowed due to electrostatic repulsions: recent studies
using hydrogen exchange and proteolysis have indicated a less-
ordered structure for protofilaments compared with mature
amyloid fibrils, 54,55 in agreement with their lower -sheet content
suggested by CD and ThT binding studies, as in the present
study. On the other hand, certain recovery of R-helical content
is observed at pH 7.4 at 150 and 250 mM NaCl concentrations,
which agrees with the decrease in ThT fluorescence intensity
observed at these concentrations, as commented previously.
FT-IR spectra recorded for HSA solutions under different
conditions at the beginning and the end of the incubation process
additionally corroborated ThT and CD data. Figure 4 shows
the second derivative of the HSA solutions at 65 °C atpH7.4
and pH 3.0 for different electrolyte concentrations. Following
incubation at 65 °C, a red-shift from 1652 to 1658 cm -1 (see
Figure S4 in the Supporting Information for the FT-IR spectrum
of native HSA) and a decrease in intensity of the amide I band
occur up to 50 mM NaCl at physiological conditions, which
are indicative of the presence of a certain increase of disordered
structure, as also seen by far-UV CD. For pH 3.0, this shift is
more progressive along the whole added salt concentration
range. The appearance of a well-defined peak around 1625 cm -1
points out a structural transformation to an intermolecular
hydrogen-bonded--sheet structure, 56 which is a structural
characteristic of the amyloid fibrils and is present at both pH
values. The spectra also show a high frequency component
(∼1693 cm -1 ) at these electrolyte concentrations that would
suggest the existence of a -sheet in an antiparallel configuration.
57 The decrease of the peak intensity at 1657 cm -1 and the
increase at 1625 cm -1 as the electrolyte concentration increases
from 0 to 50 mM NaCl at pH 7.4 support that the major
structural transition is from R-helix f -sheet and that
intermolecular interactions are favored at low and medium salt
concentrations, in agreement with CD data. In contrast, the
decrease in the band at 1625 cm -1 and the blue shift of the
band at 1542 cm -1 (not shown) at larger electrolyte concentrations
at this pH point out a lower -sheet configuration and a
further unordered conformation, which confirms that large
electrolyte concentrations do not facilitate amyloid formation,
in agreement with CD and ThT fluorescence data.
Tryptophanyl fluorescence data showed that when temperature
is raised to 65 °C, a reduction in fluorescence intensity and a
hypsochromic shift were observed, as noted in detail previously.
36 Nevertheless, we found that when incubation proceeds
at electrolyte concentrations larger than 100 mM NaCl at pH
7.4, a slightly lower decrease in fluorescence intensity takes
place, which agrees with the assumption that less ordered
amyloid-like fibrils are formed under these solution conditions
in coexistence with amorphous aggregates. In contrast, at acidic
pH, the decrease in fluorescence is also slightly larger as the
170

Fibrillation Process of Human Serum Albumin J. Phys. Chem. B, Vol. 113, No. 30, 2009 10527
Figure 6. TEM pictures of the different stages of the HSA fibrillation process at 25 °C at pH 7.4: (a) in the presence of 50 mM NaCl after 150 h
of incubation; (b) at pH 3.0 after 150 h of incubation in the absence of added salt; (c) at pH 7.4 at 65 °C in the presence of 150 mM NaCl after
50 h of incubation; (d) at pH 7.4 at 65 °C in the absence of added NaCl after 50 h of incubation; (e) at 65 °C at acidic pH in the presence of 100
mM after 150 h of incubation; (f) at 65 °C at acidic pH in the presence of 250 mM after 50 h of incubation.
added salt concentration increases, which denotes more important
changes in tertiary structure (see Figure 5).
In Situ Observation of Protein Structures. TEM images
were recorded at different stages during incubation under the
different conditions described above to denote the possible
differences existing between the aggregates as denoted previously.
Distinct time-dependent morphological stages can be
observed in these images. At room temperature, neither fibrils
nor other type of aggregates are detected at the beginning of
incubation. Only after some hours (24 h at pH 7.4, and 48 h at
pH 3.0, respectively) are some spherical aggregates detected.
These developed to amorphous clusters, which possess largely
helical structure in the presence of electrolyte upon longer
incubation (150 h), as seen in Figure 6a. In addition, at acidic
pH, less numerous, more elongated aggregates can also be
observed, with sizes of 20-30 nm in length and 3-4 nmin
width, and their population slightly becomes more important
as incubation proceeds and electrolyte concentration becomes
larger (Figure 6b). The formation of these types of aggregates
is characterized by a decrease of helical structure accompanied
with a slight rise in -sheet and unordered conformations at
long incubation times, as seen from CD measurements.
In contrast, when temperature is raised to 65 °C, fibril
formation is observed. Electron microscopy has pointed out that
aggregation leads first to globular species, which subsequently
convert to fibrils with a curly morphology via several structural
intermediates, as has been previously discussed in detail. 36 X-ray
diffraction data also confirms fibril formation (see Figure 7).
Two strong reflections at 4.8 Å and 11 Å are observed in the
X-ray image. The 4.8 Å meridional reflection arises from the
spacing between hydrogen-bonded individual strands in the sheet
structure that lie perpendicular to the fibril axis, and the
11 Å equatorial reflection corresponds to the intersheet spacing,
with the -sheets stacked face-to face to form the core structure
171

10528 J. Phys. Chem. B, Vol. 113, No. 30, 2009 Juárez et al.
Figure 7. X-ray diffraction pattern of HSA fibrils at pH 7.4 and
65 °C.
of protofilaments. 58,59 This indicates that fibrils possess a cross-
structure, one of the diagnostic halfmarks of amyloid structures.
Nevertheless, the presence of larger concentrations of added
electrolyte induces some changes in the structure of the resulting
aggregates. In this way, at salt concentrations larger than 100
mM, we have detected that the resulting fibrils appear to be
shorter and thicker, and coexist with amorphous material (see
Figure 6c and d for comparison). Enhanced attractive interactions
between structural intermediates such as beadlike structures
and lateral protofibril previously observed can be the origin of
these differences. Since electrolyte shields electrostatic repulsion
between charged groups in the protein and between protein
chains, the hydrophobic effect is progressively more important.
Electrostatic shielding allows fibril bonding not only between
fibril ends but also between axial sides of adjacent protofibrils
and protein molecules, resulting in short and thicker fibrils. In
particular, in the case when electrostatic shielding is so effective
that protein association occurs before getting suitable relative
orientations to give regular fibril assemblies, nonspecific aggregation
may take place, leading to the formation of amorphous
aggregates, provided that a suitable balance between interactions
is necessary to completely stabilize the -sheet structure.
On the other hand, at acidic conditions, the presence of
increasing electrolyte concentrations allows further growth of
the short protofibrils presented in solution at 48-96hofincubation
(depending on conditions), which do not develop
otherwise. The corresponding CD spectrum shows a progressive
increase in the proportion of the -structure at the expense of
the helical one during incubation. The presence of electrolyte
concentrations larger than 50 mM involves other structural
intermediates in the self-assembly process as ring-shaped
structures, as seen before (see also Figure S5 in the Supporting
Information). 36,60 Furthermore, as the added electrolyte concentration
increases, there is an increase in size and a denser
population of fibrils, in contrast to the behavior observed for
physiological conditions (see Figure 6f): From the previously
existing fibrils at low salt concentrations possessing sizes ranging
from ∼100 nm to several hundred nanometers in length and
8-11 nm in width (shorter on average than those obtained at
physiological conditions), which are composed of a seam of
-sheet structure decorated by relatively disorganized R-helical
structure as occurred for Rnase 61 or yeast Ure2p, 62 it is possible
to obtain larger and thicker curly fibrils at high electrolyte
concentrations due to electrostatic screening, quite similar to
those observed at pH 7 (Figure 6g).
Thus, it is interesting to note that a similar curly morphology
for HSA fibrils can be achieved under the different solution
conditions. Nevertheless, the rate and extent of aggregation
depends on solution conditions. On the other hand, it is worth
mentioning that, in contrast to HSA, BSA fibrillation has been
shown to be halted at the early curly stage in spite of the
enormous structural similarities with HSA, since no further
development in fibrillar structure over long incubation times
has been observed. 35 On the other hand, it was also noted that
the formation of the mature fibrils in vitro is concomitant with
gelation, as occurred in the present study. 63-65
Conclusions
HSA has the ability to assemble into amyloid-like aggregates
under different solution conditions, i.e., under both physiological
and acidic pH at elevated temperature and different ionic
strengths. However, the fibrillation process is strongly influenced
by the latter parameter. In this way, we have analyzed the HSA
fibrillation process and its kinetics by ThT fluorescence spectroscopy.
We observed that, at physiological pH, fibrillation is
progressively faster and more efficient in the presence of up to
50 mM NaCl due to electrostatic shielding. However, at higher
salt concentrations, an excessive shielding and further enhancement
of the solution hydrophobicity due to salt excess occurs,
which induces a reduction of the ThT fluorescence by (a)
decreasing the fraction of formed fibrils due to an unsuitable
energy balance to establish effective intermolecular interactions,
which favors random aggregation; (b) favoring side-to-side
adsorption between protein molecules and fibrils, which leads
to the formation fibril bundling, as observed by TEM; or (c)
blocking the ThT binding sites on the fibrils due to a reduction
in exposed surface area. Thus, there is a change in the energy
landscape of the fibrillation process. CD, FT-IR, and Trp
fluorescence spectra seem to confirm this picture by monitoring
the structural changes in both tertiary and secondary structure
along the fibrillation process. In this way, a reduction in -sheet
structure at large salt concentrations is also confirmed from the
analysis of far-UV CD spectra. In contrast, under acidic
conditions, a progressive enhancement of HSA fibrillation is
observed as the electrolyte concentration in solution increases.
Both the distinct ionization state and the different initial
structural state of the protein at acidic conditions (expanded
E-state) may be the origin of this behavior. On the other hand,
we have noted that the fibrillation process of HSA does not
show a lag-phase, except at acidic pH in the absence of added
salt. This involves that the initial aggregation is a downhill
process which does not require a highly organized and unstable
nucleus, with a progressive increase of the -sheet (up to 26%)
and an unordered conformation at the expense of the R-helical
conformation. Finally, the obtained fibrils show a curly morphology
and differ in length depending on solution conditions.
In fact, at acidic conditions, a progressive evolution of the
resulting fibrils can be found from a seam of -sheet structure
decorated by relatively disorganized R-helical structure to highly
structured fibrils, as found under physiological conditions, as
derived from CD and FT-IR data.
Acknowledgment. The authors thank the Ministerio de
Ciencia e Innovación for support via Project MAT-2007-61604.
The authors also thank Dr. Eugenio Vázquez for his assistance
with CD measurements.
Supporting Information Available: Figures showing a
difference absorbance spectrum of Congo Red, time evolution
172

Fibrillation Process of Human Serum Albumin J. Phys. Chem. B, Vol. 113, No. 30, 2009 10529
of ThT fluorescence, and FT-IR spectra of native HSA. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
(1) Van der Linden, E.; Venema, P. Curr. Opin. Colloid Sci. 2007,
12, 158.
(2) Shahi, P.; Sharma, R.; Sanger, S.; Kumar, I.; Jolly, R. S.
Biochemistry 2007, 46, 7365.
(3) Dobson, C. M. Nature 2003, 426, 884.
(4) Fändrich, M. Cell. Mol. Life Sci. 2007, 64, 2066.
(5) Wang, W. Int. J. Pharm. 2005, 289, 1.
(6) Gazit, E. FEBS J. 2007, 274, 317.
(7) Chiti, F.; Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi,
G.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3590.
(8) Cherny, I.; Gazit, E. Angew. Chem., Int. Ed. 2008, 48, 4062.
(9) Gras, S. L.; Tickler, A. K.; Squieres, A. M.; Devlin, G. L.; Horton,
M. A.; Dobson, C. M.; MacPhee, C. E. Biomaterials 2008, 29, 1553.
(10) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329.
(11) Thirumulai, D.; Klimov, D. K.; Dima, R. I. Curr. Opin. Struct.
Biol. 2003, 13, 146.
(12) Qu, Y.; Bolen, C. L.; Bolen, D. W. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 9268.
(13) Chiti, F.; Taddei, N.; Bucciantini, M.; White, P.; Ramponi, G.;
Dobson, C. M. EMBO J. 2000, 19, 1441.
(14) Niraula, T. N.; Haraoka, K.; Ando, Y.; Li, H.; Yamada, H.; Akasaka,
K. J. Mol. Biol. 2002, 320, 333.
(15) Krebs, M. R.; Wilkins, D. K.; Chung, E. W.; Pikeathly, M. C.;
Chamberlain, A. K.; Zurdo, J.; Robinson, C. V.; Dobson, C. M. J. Mol.
Biol. 2000, 300, 541.
(16) Moraitakis, G.; Goodfellow, J. M. Biophys. J. 2003, 84, 2149.
(17) De Felice, F. G.; Vieira, M. N.; Meirelles, M. N.; Morozova-Roche,
L. A.; Dobson, C. M.; Ferreira, S. T. FASEB J. 2004, 18, 1099.
(18) Ferrao-Gonzales, A. D.; Souto, S. O.; Silva, J. L.; Foguel, D. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 6445.
(19) Khurana, R.; Gillespie, J. R.; Talapatra, A.; Minert, L. J.; Ionescu-
Zanetti, C.; Mollet, I.; Fink, A. L. Biochemistry 2001, 40, 3525.
(20) Zurdo, J.; Guijarro, J. I.; Jiménez, J. L.; Saibil, H. R.; Dobson,
C. M. J. Mol. Biol. 2001, 311, 325.
(21) Uversky, V. N.; Fink, A. L. Biochim. Biophys. Acta 2004, 1698,
131.
(22) Plakoutsi, G.; Taddei, N.; Stefani, M.; Chiti, F. J. Biol. Chem. 2004,
279, 14111.
(23) Eakin, C. M.; Attenello, F. J.; Morgan, C. J.; Miranker, A. D.
Biochemistry 2004, 35, 6470.
(24) Fändrich, M. C.; Dobson, C. M. EMBO J. 2002, 21, 5682.
(25) Timasheff, S. N. Annu. ReV. Biophys. Biomol. Struct. 1993, 22,
67.
(26) Hoyer, W.; Anthony, T.; Cherny, D.; Heim, G.; Jovin, T. M.;
Subramaniam, V. J. Mol. Biol. 2002, 322, 383.
(27) Chiti, F.; Calamai, M.; Taddei, N.; Stefani, M.; Ramponi, G.;
Dobson, C. M. Proc. Natl. Acad. Sci. 2002, 99, 16419.
(28) Taboada, P.; Barbosa, S.; Castro, E.; Mosquera, V. J. Phys. Chem.
B 2006, 110, 20733.
(29) Taboada, P.; Barbosa, S.; Castro, E.; Gutiérrez-Pichel, M.; Mosquera,
V. Chem. Phys. 2007, 340, 59.
(30) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein
Sci. 1995, 4, 2411.
(31) Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287, 252.
(32) Holm, N. K.; Jespersen, S. K.; Thomassen, L. V.; Wolff, T. Y.;
Sehgal, P.; Thomsen, L. A.; Christiansen, G.; Andersen, C. B.; Knudsen,
A. D.; Otzen, D. E. Biochim. Biophys. Acta 2007, 1774, 1128.
(33) Pearce, F. G.; Mackintosh, S. H.; Gerrard, J. A. J. Agric. Food
Chem. 2007, 55, 318.
(34) Gorinstein, S.; Caspi, A.; Rosen, A.; Goshev, I.; Zemser, M.; Weisz,
M.; Añon, M. C.; Libman, I.; Lerner, H. T.; Trakhtenberg, S. J. Peptide
Res. 2002, 59, 71.
(35) Juárez, J.; Taboada, P.; Mosquera, V. Biophys. J. 2009, 96, 2353.
(36) Sagis, L. M. C.; Veerman, C.; Van der Linden, E. Langmuir 2004,
20, 924.
(37) Shaw, A. K.; Pal, S. K. J. Photochem. Photobiol. B 2008, 90, 69.
(38) Muzammil, S.; Kumar, Y.; Tayyal, S. Eur. J. Biochem. 1999, 266,
26.
(39) Pereira, L. G. C.; Théodoly, O.; Blanch, H. W.; Radke, C. J.
Langmuir 2003, 19, 2349.
(40) Farruggia, B.; Rodriguez, F.; Rigatuso, R.; Fidelio, G.; Picó, G. J.
Protein Chem. 2001, 20, 81.
(41) Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Brigth,
F. V. Biophys. J. 1998, 75, 1084.
(42) Tavirani, M. R.; Moghaddamnia, S. H.; Ranjbar, B.; Amani, M.;
Marashi, S. A. J. Biochem. Mol. Biol. 2006, 39, 530.
(43) Rader, A. J.; Hespenheide, B. M.; Kuhn, K. A.; Thorpe, M. F. Proc.
Natl. Acad. Sci U.S.A. 2002, 99, 3540.
(44) Ferrone, F. Methods Enzymol. 1999, 309, 256.
(45) Lin, M.-S.; Chen, L.-Y.; Tsai, H.-T.; Wang, S. S.-S.; Chang, Y.;
Higuchi, A.; Chen, W.-Y. Langmuir 2008, 24, 5802.
(46) Hills, R. D.; Brooks, C. L. J. Mol. Biol. 2007, 368, 894.
(47) Hong, Y.; Pritzker, M. D.; Legge, R. L.; Chen, P. Colloids Surf.,
B 2005, 46, 152.
(48) Fujiwara, S.; Matsumoto, F.; Yonezawa, Y. J. Mol. Biol. 2003,
331, 21.
(49) Sikkink, L. A.; Ramírez-Alvarado, M. Biophys. Chem. 2008, 135,
25.
(50) Zheng, J.; Jang, H.; Nussinov, R. Biochemistry 2008, 47, 2497.
(51) Cerda-Costa, N.; Esteras-Chopo, A.; Aviles, F. X.; Serrano, L.;
Villegas, V. J. Mol. Biol. 2007, 366, 1351.
(52) Plakoutsi, G.; Bemporad, F.; Calamai, M.; Taddei, N.; Dobson,
C. M.; Chiti, F. J. Mol. Biol. 2005, 351, 910.
(53) Sreerama, N.; Woody, R. W. Protein Sci. 2003, 12, 384.
(54) Kheterpal, I.; Chen, M.; Cook, K. D.; Wetzel, R. J. Mol. Biol. 2006,
361, 785.
(55) Myers, S. L.; Thomson, N. H.; Radford, S. E.; Ashcroft, A. E. Rapid
Commun. Mass Spectrom. 2006, 20, 1628.
(56) Casal, H. L.; Kohler, U.; Mantsch, H. H. Biochim. Biophys. Acta
1988, 957, 11.
(57) Fabian, H.; Choo, L.-P.; Szendrei, G. I.; Jackson, M.; Halliday,
W. C.; Otvos, L., Jr.; Mantsch, H. H. Appl. Spectrosc. 1993, 47, 1513.
(58) Sunde, M.; Blake, C. AdV. Protein Chem. 1997, 50, 123.
(59) Bouchard, M.; Zurdo, J.; Nettleton, E. J.; Dobson, C. M.; Robinson,
C. V. Protein Sci. 2000, 9, 1960.
(60) Sibley, S. P.; Sosinsky, K.; Gulian, L. E.; Gibbs, E. J.; Pasternack,
R. F. Biochemistry 2008, 47, 2858.
(61) Sambashivan, S.; Liu, M. R.; Sawaya, M. R.; Gingery, M.;
Eisenberg, D. Nature 2005, 437, 266.
(62) Ranson, N.; Stromer, T.; Bousset, L.; Melki, R.; Serpell, R. C.
Protein Sci. 2006, 15, 2481.
(63) Ramírez-Alvarado, M.; Merkel, J. S.; Regan, L. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 8979.
(64) Damaschun, G.; Damaschun, H.; Fabian, H.; Gast, K.; Krober, R.;
Wieske, M.; et al. Proteins: Struct., Funct., Genet. 2000, 39, 204.
(65) Goda, S.; Takano, K.; Yamagata, Y.; Nagata, R.; Akutsu, H.; Maki,
S.; et al. Protein Sci. 2000, 9, 369–375.
JP902224D
173

Additional Supra-Self-Assembly of Human Serum Albumin under Amyloid-Like-Forming
Solution Conditions
Introduction
Josué Juárez, † Pablo Taboada,* ,† Sonia Goy-López, † Adriana Cambón, †
Marie-Beatrice Madec, ‡ Stephen G. Yeates, ‡ and Víctor Mosquera †
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada,
Facultad de Física, UniVersidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain, and
Organic Materials InnoVation Center, School of Chemistry, UniVersity of Manchester, Manchester M13 9PL,
United Kingdom
ReceiVed: May 5, 2009; ReVised Manuscript ReceiVed: June 22, 2009
Protein aggregation has a multitude of consequences ranging from affecting protein expression to its implication
in different diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid
fibrils, structures associated with diseases such as Alzheimer’s disease. These fibrils can further associate in
other more complex structures such as fibrillar gels, plaques, or spherulitic structures. In the present work,
we describe the physical and structural properties of additional supraself-assembled structures of human serum
albumin under solution conditions in which amyloid-like fibrils are formed. We have detected the formation
of ordered aggregates of amyloid fibrils, i.e., spherulites which possess a radial arrangement of the fibrils
around a disorganized protein core and sizes of several micrometers by means of polarized optical microscopy,
laser confocal microscopy, and transmission electron microscopy. These spherulites are detected both in solution
and embedded in an isotropic matrix of fibrillar gels. In this regard, we have also noted the formation of
protein gels when the protein concentration and/or ionic strength exceds a threshold value (the gelation point)
as observed by rheometry. Fibrillar gels are formed through intermolecular nonspecific association of amyloid
fibrils at a pH far away from the isolectric point of the protein where protein molecules seem to display a
“solid-like” behavior due to the existence of non-DLVO (Derjaguin-Landau-Verwey-Overbeck) intermolecular
repulsive forces. As the solution ionic strength increases, a coarsening of this type of gel is observed
by environmental scanning microscopy. In contrast, at pH close to the protein isoelectric point, particulate
gels are formed due to a faster aggregation process, which does not allow substantial structural reorganization
to enable the formation of ordered structures. This behavior also additionally corroborates that the existence
of particulates might also be a generic property of all polypeptide chains as amyloid fibril formation under
suitable conditions.
A number of neurodegenerative diseases have been related
to protein aggregation and deposition in the form of amyloid
fibrils, such as type II diabetes, Alzehimer’s, Parkinson’s,
Huntington’s, and prion diseases amongst others. 1-6 A wide
range of proteins is known to misfold and aggregate in mildly
or hard denaturating conditions as amyloid fibrils provided that
the ability to fibrillate is independent of the original native
structure of the protein, whose amino acid sequence primarily
appears to play a key role in terms of filament arrangement, 7
fibrillation kinetics, 8 and overall yield and stability of the
fibrils. 9,10 These fibrils have been found to be based on a
common structural motif consisting of continuous intermolecular
-sheets which run along the fibril axis such that the individual
-strands are perpendicular to the fibril axis. 11
Under certain conditions, protein fibrils can further associate
either nonspecifically such as in fibrillar gels or plaques or in a
more ordered fashion such as in spherulites. 12-18 It is known
that fibrillar gels are formed by protein aggregation if the protein
concentration exceeds a given critical value under conditions
* Author to whom correspondence should be addressed. E-mail:
pablo.taboada@usc.es.Phone:0034981563100,Ext.14042.Fax:0034981520676.
† Universidad de Santiago de Compostela.
‡ University of Manchester.
J. Phys. Chem. B 2009, 113, 12391–12399 12391
where protein molecules are partially denaturated (as at high
temperatures) and electrically charged (this is, far away from
their isolectric point) upon incubation, i.e., under conditions
where fibrillation can occur. Nevertheless, the correspondence
between fine strand gels and amyloid fibril structure was
established recently. 19,20 In contrast, by decreasing the intermolecular
repulsion through shifting the solution pH to values
near the protein isoelectric point, or by increasing the solution
ionic strength the gel networks are comprised of particulate
aggregates.
On the other hand, for systems where fibrillar protein aggregates
and gels are formed, the strands can further associate in a more
ordered fashion such as spherulitic structures. These are characterized
by the presence of anisotropic material ordered in a spherically
symmetric way. This gives rise to the appearance of a Maltese
cross extinction pattern when these structures are studied under a
polarized light microscope, much in the same way as spherulites
formed by synthetic polymers, 21,22 natural polymers, and biopolymers.
23-25 In solutions in Vitro, spherulites have been reported for
different synthetic and natural peptides, 17,26-30 and have also usually
been associated to the amyloid-like formation pathways of
several proteins such as -lactoglobulin, 20,31,32 R-L-iduronidase, 33
lysozyme, 18 and insulin. 16,34 Spherulites were also found in ViVo
in Alzheimer’s disease, Gerstmann-Straulsler-Scheinker dis-
10.1021/jp904167e CCC: $40.75 © 2009 American Chemical Society
Published on Web 08/14/2009
175

12392 J. Phys. Chem. B, Vol. 113, No. 36, 2009 Juárez et al.
ease, Down’s syndrome, 35 and in tumors. 36,37 Thus, the formation
of fibrillar structures may not be the end of the aggregation
process, and proper knowledge of the nature and structure of
these supramolecular assemblies may have important implications
in different fields such as medicine, 3,4,35-37 food industry, 38,39
or biomaterials. 40
In recent reports, 41,42 we have shown that partially destabilized
human serum albumin (HSA) molecules form amyloid-like
fibrils under different solution conditions. These fibrils feature
the structural characteristics for amyloids: X-ray diffraction
patterns, affinity to Congo Red and Thioflavin T dyes, birefringence,
and high stability. Nevertheless, different fibrillation
pathways, fibrillation kinetics, and structural intermediates and
morphological differences in the resulting fibrils were observed
depending on the incubation conditions. 42
In this paper, we extend our previous studies in order to
explore the solution conditions which enable the formation of
different supramolecular assemblies of HSA amyloid-like fibers,
and the structure of these resulting assemblies. To do that,
extensive incubation of HSA solutions of pH 2.5, 5.5, and 7.4
at elevated temperature and ionic strengths ranging from 0 to
250 mM NaCl was made, and the resulting protein assemblies
were analyzed by optical, confocal, transmission electron,
environmental scanning electron, and atomic force microscopies,
rheometry, and attenuated reflectance Fourier transform infrared
(ATR-FTIR) and fluorescence spectroscopies. We have observed
that spherulitic formation takes place under conditions where
amyloid fibrils were previously present. The spherulites possess
a radial arrangement of the protein fibrils around a disorganized
protein core and sizes of 5-50 µm. In addition, different types
of gels were also obtained upon extensive incubation under
different solution conditions, either with fibrillar or particulate
structure. Fibrillar gels that form at a pH far from the isoeletric
point of the protein display a “solid-like” behavior and are
formed through intermolecular nonspecific association of amyloid
fibrils. A coarsening of these gels occurs when the solution
ionic strength increases. When the solution pH is close to the
protein isoelectric point, particulate gels are formed. These
originate from a faster protein aggregation which does not allow
the necessary structural reorganization to enable the formation
of more ordered structures.
Materials and Methods
Materials. Human serum albumin (70024-90-7) and Thioflavin
T (ThT) were obtained from Sigma Chemical Co. and used
as received. All other chemicals were of the highest purity
available.
Preparation of HSA Solutions. Protein was used after further
purification by liquid chromatography using a Superdex 75
column equilibrated with 0.01 M phosphate. Experiments were
carried out using double distilled, deionized, and degassed water.
The buffer solutions used were glycine + HCl (I ) 0.01 M)
for pH 2.5, sodium acetate-acetic acid for pH 5.5 (I ) 0.01
M), and sodium monophosphate-sodium diphosphate for pH
7.4 (I ) 0.01 M), respectively. HSA was dissolved in each buffer
solution to a final concentration of typically 20 mg/mL if not
otherwise stated, and dialyzed extensively against buffer solution.
Protein concentration was determined spectrophotometrically,
using a molar absorption coefficient of 35219 M-1 cm-1 at 280 nm. 43 Prior to incubation, solutions were filtered through
a 0.2 µm filter into sterile test tubes. Samples were incubated
at a specified temperature (65 °C) for up to 15 days in a refluxed
reactor if not otherwise stated. Samples were taken out at
intervals and stored on ice before adding ThT.
Polarized Optical Microscopy (POM). After incubation,
aliquots of protein solutions were removed from the vials and
put onto microscope slides. These were immediately studied
using a Zeiss Axioplan optical microscope (Carl Zeiss Ltd.,
Welwyn Garden City, U.K.). A total magnification of either
50× or 100× was used. A polarizer and analyzer were put in
fixed positions, orthogonal to one another for the cross-polarizer
imaging. Images were taken using a Kodak digitial camera
mounted on the top of the microscope. The size of the
spherulites seen in the images was determined by calibrating
with a scale bar of known dimensions and thereby quantifying
the spatial resolution per pixel image.
Laser Confocal Microscopy. Confocal microscopy was
performed on a Leica TCS SP2 confocal system mounted on a
Leica DM-IRE2 upright microscope, using a 40× objective. For
fluorescence measurements, Thioflavin T was added to spherulitic
solutions and excited using the 458 nm line of an argon
ion laser. Simultaneously, transmission images were obtained
with crossed polarizers in place using the 633 nm line of a
He-Ne laser.
Thioflavin T Spectroscopy. Protein and ThT were dissolved
in proper buffer at a final protein-dye molar ratio of 50:1.
Samples were continuously stirred during measurements. Fluorescence
was measured in a Cary Eclipse fluorescence spectrophotometer
equipped with a temperature control device and
a multicell sample holder (Varian Instruments Inc.). Excitation
and emission wavelengths were 450 and 482 nm, respectively.
All intensities were background-corrected for the ThT fluorescence
in the respective solvent without the protein. Data from
at least three different separate experiments were averaged and
normalized on a scale from 0 to 1.
Attenuated Total Reflectance Fourier Transform Infrared
Spectroscopy (ATR-FTIR). ATR-FTIR spectra of HSA in
aqueous solutions were determined by using a FTIR spectrometer
(model IFS-66v from Bruker) equipped with a horizontal
ZnS ATR accessory. The spectra were obtained at a resolution
of2cm -1 , and generally 200 scans were accumulated to get a
reasonable signal-to-noise ratio. Solvent spectra were also
examined using the same accessory and instrument conditions.
Each different sample spectrum was obtained by digitally
subtracting the solvent spectrum from the corresponding sample
spectrum. Each sample solution was repeated three times to
ensure reproducibility and averaged to produce a single spectrum.
Transmission Electron Microscopy (TEM). Suspensions of
HSA were applied to carbon-coated copper grids, blotted,
washed, negatively stained with 2% (w/v) phosphotungstic acid,
air-dried, and then examined with a Phillips CM-12 transmission
electron microscope operating at an accelerating voltage of 120
kV. Samples were diluted between 20-200-fold and sonicated
when needed prior to deposition on the grids.
Environmental Scanning Electron Microscopy (ESEM).
Suspensions of HSA were applied to glass-coated stainless steel
grids, blotted, washed, air-dried, and then examined with an
LEO-435 VP scanning electron microscope (Leica Mycrosystems,
Cambridge, U.K.) operating at an accelerating voltage of
20 kV. Samples were diluted when needed prior to deposition
on the grids. Samples were left to equilibrate at 2 °C. A few
drops of distilled and deionized water were place around the
sample. The chamber was flooded repeatedly with water vapor
and the pressure subsequently reduced to ∼5 Torr. Further
decreases in pressure led to evaporation of water and drying of
the samples.
Atomic Force Microscopy (AFM). AFM images were
recorded in the tapping mode by using an AFM XE100
176

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12393
instrument (PSIA Inc., Seoul, Korea) equipped with a rigid
silicon cantilever of nominal spring constant of about 40 mN/m
and resonant frequency of 325 kHz. Immediately after incubation,
protein samples were diluted 20-400 times onto freshly
cleaved muscovite mica (from Sigma Chemical Co.) attached
to a magnetic steel disk which served as a sample holder. The
abrupt dilution of the samples immediately quenches the
concentration-dependent aggregation process. The AFM samples
were dried in air or under nitrogen flow when required. Control
samples (freshly cleaved mica, mica and buffer solution) were
also investigated with AFM to exclude possible artifacts.
Topography and phase-shift data were collected in the trace and
retrace direction of the raster, respectively. The offset point was
adapted accordingly to the roughness of the sample. The scan
rate was tuned proportionally to the area scanned and kept within
0.35-2 Hz range.
Rheometry. The rheological properties of the samples were
determined using a Bohlin CS10 rheometer with water bath
temperature control. Couette geometry (bob, 24.5 mm in
diameter, 27 mm in height; cup, 26.5 mm in diameter, 29 mm
in height) was used, with a 2.5 cm 3 sample being added to the
cup in the mobile state. Samples of very high modulus were
investigated using cone-and-plate geometry (diameter 40 mm,
angle 4°). A solvent trap maintained a water-saturated atmosphere
around the cells. Frequency scans of storage (G′) and
loss (G′′) moduli were recorded for selected protein concentrations
and temperatures with the instrument in the oscillatory
shear mode and with the strain amplitude (A) maintained at a
low value (A < 0.5%) by means of the autostress facility of the
Bohlin software. This ensured that measurements of G′ and G′′
were in the linear viscoelastic region. Measurements on solutions
of low modulus (G′ ) 1-10 Pa) which fell outside the range
for satisfactory autostress feedback were rejected.
Results and Discussion
Human serum albumin consists of 585 amino acids in a single
polypeptide chain, with a globular structure composed of three
main domains that are loosely joined together through physical
forces and six subdomains that are wrapped by disulfide bonds.
The protein contains 17 disulfide bridges and one free SH group.
Most of the HSA sequence (>60%) is arranged in R-helix
structure, with the subsequent tightening of its structure through
intramolecular interactions such as hydrogen bonds. Nevertheless,
amyloid-like formation of HSA has been reported under
different solution conditions. 41,42 HSA fibril formation usually
takes place in the absence of a lag phase, and the resulting fibrils
share the common structural features of “true” amyloid fibrils,
although their final morphology depends on the incubation
conditions. 42
Spherulite Formation and Characterization. The propensity
for spherulite formation was investigated by using optical
and confocal microscopy at pH 2.5, 5.5, and 7.4 at 65 °C upon
incubation in the presence of NaCl in the range 0-250 mM.
We have observed that spherulitic structures are found under
conditions where HSA fibrils were previously detected, 42 that
is, at physiological pH and under acidic conditions in the
presence of the highest added salt concentrations (100-250 mM
NaCl). Although data were not collected quantitatively, the
highest density of spherulites was observed at pH 7.4 in the
presence of 50 mM NaCl, and at acidic pH in the presence of
250 mM. This agrees with the maximum efficiency of fibril
formation previously reported. 42 In this regard, it has been
previously found41 that at both physiological pH and temperature
fibril formation is disfavored at high ionic strengths due to an
excesive shielding of repulsive electrostatic forces, which
involves an increase in rapid random protein aggregation. In
contrast, under acidic conditions fibril formation is continuosly
enhanced as the added salt concentration increases. This
difference has been assigned to the different nature and structure
of the protein precursor states, which leads to different fibrillation
rates, fibril conversion extents, fibrillation pathways, and
structures of resulting fibers. 44-46 In addition, no differences were
observed between the spherulites obtained in the various samples
studied regardless of the incubation conditions.
Analysis of the solutions at both pH 2.5 and 7.4 by polarized
optical microscopy allowed us to observe the presence of
classical Maltese cross extinction patterns (Figure 1a) as a key
mark of the presence of spherulites. This type of pattern was
not observed at pH 5.5. The observed spherulites possess sizes
ranging from 5 to 50 µm. In addition, the test tubes when placed
between crossed polarizers showed birefrigence from spherulites
in the samples (not shown). Spherulitic structures are detected
both in solution prior to gelification and embedded in an
isotropic gel matrix after gelification. It should also be noted
that nonspherulitic areas of the sample, which may contain fibrils
not assembled into spherulites, are not visibly birefringent,
indicating that polypeptide chains in this region are unoriented.
Similar spherulitic occurrences in protein gels were also found,
for example, for whey proteins, 47 -lactoglobulin, 29,30 and
insulin. 32,33
To confirm that the spherulites contain amyloid fibrils, they
were imaged by confocal microscopy using ThT as the
fluorescent dye, whose fluorescence is known to increase
markedly upong binding to amyloid fibrils. 48 In confocal mode,
the fluorescence emission was observed to come from the
spherulites (Figure 1b), which confirms they are composed of
amyloid fibers. 49 Confocal microscopy in transmission mode
resulted in an image similar to that obtained with an optical
microscope (Figure 1c), clearly displaying the presence of
spherulites as Maltese cross patterns of comparable sizes to those
observed by optical miscroscopy. The nonordered and nonfluorescent
core is probably due to nonpenetration of ThT. As this
core is present even after extensive incubation in the presence
of the dye, a more feasible explanation is the absence of
significant amounts of amyloid material in this core, that is, it
is formed before spherulitic growth takes place due to an
interplay between amyloid fibrillation and protein random
aggregation. 16 Aliquots of spherulitic solutions were also added
to solutions containing ThT. The enhanced fluorescence relative
to solutions of ThT and ThT containing freshly dissolved HSA
additionally proved the presence of amyloid structures forming
the spherulites (see Figure 1d).
To reveal the orientation of the fibrils within the spherulites,
insertion of a waveplate at 45° angle to the cross polars provided
confirmation of this radial arrangement (see inset in Figure 1a).
The four quadrants in the spherulites are now colored. The
appearance of the blue color indicates that the fast optical axis
of the spherulites is aligned with the fast optical axis of the
wave retardation plate. 34 The slow direction within the spherulite
is the radial direction and, therefore, indicates that the fibrils
lie radially within the spherulites, as seen previously for other
proteins such as -lactoglobulin, 20,31,32 R-L-iduronidase, 33
lysozyme, 18 and insulin. 16,34 Figure 1e shows that out from the
core the radially oriented internal structure appears to be strands
of fibril bundles (because individual fibrils are too narrow to
be seen optically) with slight curvature to their orientation and
without obvious branching. This is particularly viewable at the
177

12394 J. Phys. Chem. B, Vol. 113, No. 36, 2009 Juárez et al.
Figure 1. (a) Optical microscopy images of grown spherulites of HSA at pH 7.4 in the absence of added NaCl at 65 °C after 72 h of incubation
under cross-polarizers. The inset indicates the radial arrangement of fibrils through insertion of a waveplate at 45° between the polarizers. (b)
Fluorescence image in the confocal mode of spherulites using ThT as a probe. (c) Confocal image in the transmission mode of spherulites between
cross-polarizers. (d) ThT fluorescence upon incubation in a solution without HSA, containing native HSA and containing spherulites. (e) Optical
microscopy image of HSA spherulites without polarizers. Scale bars are (a) 50, (b) 200, and (c and e) 100 µm.
outer edges of the spherulites, where the fibril bundles are
slightly separated from one another.
The spherical morphology of spherulites was also observed
by ESEM (see Figure 2), with sizes similar to those derived
from optical and confocal microscopy. Their surface appears
to have a texture on the length scale of several micrometers
but shows no obvious orientational order. Some cracks on the
particle surface appeared as a consequence of the dehydration
process in ESEM. Unfortunately, we could not observe if the
spherulites formed display a radial internal order, since internal
cracking of spherulitic structures was not achieved.
Direct measurement of spherulitic solutions was not posible
by TEM provided that a film was formed on the grids which
do not allow visualization. In this way, dilution and sonication
of the spherulitic solutions were performed in order to investigate
the samples by TEM. This treatment resulted in the
disruption of the spherulites and the appearance of amyloidlike
fibril structures, as seen in Figure 2d. This fact further
confirms that spherulites are mainly composed of amyloid fibrils.
Gel Formation and Characterization. Prolonged incubation
of solutions containing 0.3 mM HSA (or 0.8 mM where
corresponds) at either pH 7.4, 5.5 or 2.5 at 65 °C in the presence
of different amounts of added salt leads to the formation of
gels. In this work, we have only analyzed solution conditions
previously studied for amyloid fibril formation. 41,42 Significant
differences were observed in the association properties of the
different protein solutions. In this way, we have observed that
at pH 7.4 and 5.5 gels were formed at electrolyte concentrations
larger than 20 mM with gelation times decreasing from several
days to hours as the solution ionic strength increases (or the
protein concentration rises). On the other hand, under acidic
conditions gels were only formed at the highest electrolyte
concentration analyzed, 250 mM NaCl at 0.3 mM HSA. In
general, it seems that gel formation is favored by the screening
of electrostatic interactions between protein molecules, which
favors protein aggregation. 45,46 Nevertheless, protein interactions
at acidic pH are weaker than those at physiological pH due to
the initial different conformational state of the protein (Expanded-E
state under acidic conditions), which imparts a larger
solubility to the acid-denaturated protein molecules as a result
of electrostatic repulsion between them due to an increase of
surface-charged groups.
Figure 3a shows cure curves (G′, the storage modulus, versus
time) for gelling HSA solutions at pH 7.4, 5.5, and 2.5 in the
presence of 100 mM NaCl at 65 °C. To follow the protein
gelation in a suitable time scale for the experiment, we increased
the protein concentration up to 0.8 mM. The obtained cure data
are typical of a sol-gel transition of biopolymer solutions: The
elastic component of the shear modulus starts small but
undergoes a sudden increase at a critical time and, then,
ultimately almost levels off at longer times. For pH 7.4 and
2.5, the behavior is similar to that expected for a strongly gelling
178

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12395
Figure 2. (a-c) ESEM images of different HSA spherulites incubated at pH 7.4 at 65 °C in the absence of added electrolyte along dehydration.
Progressive dehydration allows a better visualization of the spherulites. In part c, some surface cracking can be observed. The scale bars are 200,
100, and 50 µm in parts a, b, and c, respectively. (d) TEM image of HSA fibrils obtained after disruption of grown spherulites by extensive
sonication.
Figure 3. (a) G′ versus time (cure data) for a 0.8 mM HSA solution
heated at 65 °C atpH(9) 7.4, (b) 2.5, and (4) 5.5 in the presence of
100 mM NaCl. (b) Time evolution of storage (G′, O) and loss (G′′, 9)
moduli of a 0.8 mM HSA solution heated at 65 °C and pH 7.4 in the
presence of 100 mM NaCl.
biopolymer solution, with the divergence between G′ and G′′
becoming larger with time and G′′ < G′. Another feature of
these cure curves is the “dip-down” in the modulus at long times,
which may be indicative of slippage of the gel samples. 13
Interestingly, G′, though small initially at both pHs, was larger
than G′′ (the loss modulus) even before the gelling point (see
Figure 3b as an example). This “solid-like” behavior of the
starting protein solutions has been ascribed to colloid-like
Figure 4. Frequency depedence of the storage (G′, closed symbols)
and loss (G′′, open symbols) moduli of a 0.8 mM HSA solution at pH
(b) 7.4, (9) 2.5, and (2) 5.5 in the presence of 100 mM NaCl at 65
°C.
structuring in the pregel solution due to nonclassical DLVO
forces. 50,51 This is indicative of a different self-assembly
mechanism and/or kinetics if compared to what was observed
at pH 5.5, for which G′′ > G′ at short incubation times. At the
latter pH, faster aggregation takes place since enhanced protein/
protein interactions are favored due to the proximity of the
solution pH to the protein isoelectric point. It has been pointed
out that rapid formation of an appreciable amount of intermolecular
-sheets favored by protein/protein interactions 52 involves
very compact structures which precipitate, thereby limiting the
amount of protein available to form protein/water networks
necessary for robust gel formation 53 as in the present case. This
fact is also corroborated comparing the lower G′ values at pH
5.5 to those obtained at pH 7.4 and 2.5.
Figure 4 shows the corresponding frequency spectrum of G′
and G′′ for the gels after completion of the cure experiment.
Samples at pH 2.5 and 7.4 show typical gel spectra. G′ is
179

12396 J. Phys. Chem. B, Vol. 113, No. 36, 2009 Juárez et al.
Figure 5. (a and b) ESEM and (c) AFM images showing progressive coarsening of fibril strands of HSA gels after incubation at 65 °C atpH7.4
in the presence of 20, 100, and 250 mM NaCl, respectively (scale bars in parts a and b are 10 and 5 µm, respectively; AFM image size is 5 µm
× 5 µm). (d) TEM and (e) AFM images of fibrils from coarsed HSA gels after disruption by dilution and sonication at physiological pH (AFM
image size is 5 µm × 5 µm). (f and h) AFM and (g) ESEM images of particulate gels of HSA at pH 5.5 after incubation at 65 °C in the presence
of 20 (f and g) and 150 mM NaCl (h) (scale bar in part g is 2 µm; AFM image size is 5 µm × 5 µm).
essentially frequency-independent, so we can assert that the
storage moduli will have a finite value at very long times (low
frequencies); i.e., it confirms the solid-like properties consistent
with the development of a network, as previously commented.
On the other hand, G′ values at physiological pH are larger than
those at acidic pH, which denotes the formation of a stiffer gel.
This is also supported by the essentially frequency-independent
behavior of G′′ at pH 7.4. In contrast, under acidic conditions,
a slight minimum in G′′ can be observed, which becomes much
more intense at pH 5.5. This minimum at pH 5.5 can be assigned
to molecular packing, 54 and is in agreement with the lower G′
values, the frequency-dependent behavior, and the particulate
structure of the gels formed at this pH, as seen below.
Figure 5 shows ESEM and AFM images of the gels formed
at pH 7.4, 5.5, and 2.5 at different ionic strengths. For
physiological conditions after incubation at 65 °C in the presence
of 20 mM NaCl, a fibrillar gel formed by fine strands can be
observed (Figure 5a). These strands are formed by interconnected
amyloid-like fibrils as denoted by the cross- pattern
derived from XRD analysis (see Figure S1 in the Supporting
Information), and in agreement with the rheology data. Moreover,
upon strong dilution and sonication the strands dissociate
into fibrils which showed a predominant curly morphology, as
observed previously for HSA 42 and other proteins such as
2 microglobulin, 55 R-crystallin, 56 or bovine serum albumin
(BSA). 57 Nevertheless, some mature straight fibrils and ribbonlike
structures could also be observed (see Figure S2 in the
Supporting Information).
When the ionic strength is increased at pH 7.4 up to 250
mM NaCl, a coarsening of the gel is progressively observed,
180

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12397
with the presence of relatively long, large, and thick aggregates
which seem to be composed, at least partially, of globular
aggregates (Figure 5b and c). Additionally, the size of the
elementary particles appeared to be larger than those formed at
lower added salt concentrations. A similar picture was observed
for the gel formed at acidic pH (not shown). Therefore, a
transition from fine-stranded to coarse gels with increasing ionic
strength seems to be caused mainly by kinetic effects without
accompanying fundamental changes in aggregation mechanisms.
58 After extensive dilution and sonication, rupture of these
types of aggregates in a variety of fibrillar structures rather than
in smaller spherical aggregates was observed by TEM and AFM
(see Figure 5d and e), as seen in previous studies. 59 The length
of the fibrils varies between 0.1 and 1-2 µm with a thickness
of 15-30 nm.
In contrast, at pH 5.5 a network of quasipherical protein
aggregates surrounded by liquid is formed; i.e., we observed a
particulate gel (see Figure 5f-h). The particle size varies
between ∼300 and ∼500 nm, which increases as the ionic
strength increases. This leads to fused long and thick aggregates
possibily composed of several particulates. In this regard, it has
been speculated that intermolecular disulfide bonding is involved
in connecting protein molecules within the particulates and that
the connections among them is a nonspecific physical crosslinking
without any specific connective sites. 58,60 ESEM also
indicates that an amount of residual monomeric protein was
left in the solutions (Figure 5g) and deposited between the
particulates. This gives the appearance of connected particulates
and, sometimes, apparently amorphous films. As commented
above, shifting the pH toward the isoelectric point decreases
the intermolecular electrostatic repulsion. This implies that
aggregation becomes faster, preventing the formation of highly
ordered nanostructures such as amyloid-like fibrils, but globular
shapes are formed.
Thus, it seems that the gelation of HSA is also pH-dependent
and the structures which form the gel depend on the solution
conditions. In this regard, in a recent report the capability of
several proteins such as -lactoglobulin, BSA, insulin, and
lysozyme among others to form either fibrillar or particulate
gels under partially denaturing conditions but at different pHs
has been confirmed. 61 This suggests that the formation of
particulates can be a generic property of all polypeptide chains
like amyloid fibrillation is. In this way, the results reported in
this work support this view.
Finally, in order to investigate the internal structure of the
fibrillar and particulate gels, attenuated total reflectance Fourier
transform infrared spectroscopy (ATR-FTIR) measurements
were made. All spectra were well resolved with clearly
distinguishable secondary structure signatures. In this way,
before incubation two major bands peaks in the second
derivative IR spectra in the spectral region of interest were
observed at pH 7.4: the amide I band at 1652 cm -1 and the
amide II band at 1544 cm -1 . This indicates the predominant
structural contribution of major R-helix and minor random coil
structures. 62,63 For the amide I band (see Figure 6a), a shoulder
at ca. 1630 cm -1 can also be observed in the second derivative
spectra, which is related to a low intramolecular -sheet content.
Additional peaks at ca. 1689 and 1514 cm -1 would correspond
to -turn and tyrosine absorption, respectively. 63 As the pH is
lowered, the remaining presence of the amide I and II bands
confirms that there is still a significant amount of R-helices,
even at the most acidic pH (see Figure 6a).
Following incubation at 65 °C and formation of the gel at
pH 7.4, a red-shift of the amide I band from 1652 to 1658 cm -1
Figure 6. Second derivative of FTIR spectra of HSA samples formed
at (a) pH 2.5, (b) pH 5.5, and (c) pH 7.4 in the presence of 50 mM
NaCl (s) before and ( ···) after incubation and gelation at 65 °C.
(1650 to 1656 cm -1 at acidic pH) and a blue-shift of the amide
II band to 1542 cm -1 (1540 cm -1 for pH 2.5) is indicative of a
certain increase of disordered structure (see Figure 6b). The
appearance of a well-defined peak around 1625 cm -1 (1624
cm -1 for pH 2.5) points to a structural transformation from an
intramolecular hydrogen-bonded -sheet to an intermolecular
hydrogen-bonded--sheet structure, 64 which is a structural
characteristic of the amyloid fibrils. The spectrum also shows
a high frequency component (∼1693 cm -1 ) that would suggest
the presence of an antiparallel -sheet. 65 In addition, a small
shoulder around 1534 cm -1 was also assigned to a -sheet. 66
In the case of particulate gels formed under incubation at 65
°C at pH 5.5, we could also observe a decrease and shift to
1657 cm -1 of the amide I band and an increase in the content
of -sheet conformation. This is indicated by the enhancement
of the intensity and further shift of the band positioned around
1626 cm -1 , in agreement with previous reports (see Figure
6c). 61,67,68 In addition, the proportion of -sheet content is lower
than that at pH 7.4 and 2.5. This corroborates that the
aggregation near the protein isoelectric point takes place faster
and nonspecifically which decreases the likehood of substantial
structural rearrangements during the aggregation process. In
contrast, amyloid fibrils and fibrillar gels resulted from partially
highly charged unfolded states, 41,42 which involve long-range
repulsion and slow aggregation occurring only when substantial
structural reorganization allows the formation of a favorable
structure, the cross- structure. In fact, when excess electrolyte
is added, aggregation becomes faster and the proportion of
-sheet structure decreases due to an enhancement of the
aggregation rates. 46
Conclusions
In this work, we have described the existence of suprafibrillar
assemblies formed by the protein human serum
181

12398 J. Phys. Chem. B, Vol. 113, No. 36, 2009 Juárez et al.
albumin under different solution conditions: spherulites and
fibrillar gels. Under conditions where amyloid-like fibrils for
this protein were previously detected, that is, upon incubation
at 65 °C at both acidic and physiological pH in the presence
of different amounts of added electrolyte, spherulite formation
was detected. Within spherulites, fibrils display a radial
arrangement around a disoganized protein core with sizes of
several micrometers, as revealed by POM and confocal
microscopy. The spherulites are detected both in solution and
embedded in an isotropic matrix of a fibrillar gel. Upon
extensive incubation under increasingly added electrolyte and/
or protein concentration, gels are formed. Under conditions
where the protein is electrically charged, fibrillar gels were
observed. In contrast, at a pH close to the protein isolectric
point, particulate gels were observed. Fibrillar gels are formed
through intermolecular nonspecific association of amyloid
fibrils at a pH far away from the isolectric point of the protein,
as observed by TEM, AFM, and ATR-FTIR. For these types
of gels, the protein molecules seem to display a “solid-like”
behavior due to the existence of non-DLVO intermolecular
repulsive forces, as observed by rheometry. As the solution
ionic strength increases, a coarsening of this type of gels is
observed as seen by AFM, with a decrease in the elastic
response. In contrast, at pH 5.5, particulate gels are present
as a consequence of a faster protein aggregation which does
not allow the necessary structural reorganization to enable
the formation of more ordered structures such as fibrils, as
observed by their shorter gelation times and the frequencydependent
behavior of the loss modulus. On the other hand,
the formation of particulate gels with HSA also confirms the
possibility that this type of structure may be a generic
property of the polypeptide chains under suitable solution
conditions.
Acknowledgment. Authors thank Ministerio de Educación
y Ciencia by project MAT-2007-61604 and Xunta de Galicia
for financial support.
Supporting Information Available: XRD fibril diffraction
pattern and TEM pictures of mature fibrils. This material is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Hady, J.; Selkoe, D. Science 2002, 297, 353–356.
(2) Dobson, C. M. Nature 2003, 426, 884–890.
(3) Stefani, M.; Dobson, C. M. J. Mol. Med. 2003, 81, 678–699.
(4) Lansbury, P. T., Jr. Nat. Med. 2004, 10, 13709–13715.
(5) Mattson, M. P. Nature 2004, 430, 631–639.
(6) Soto, C.; Estrada, L.; Castilla, J. Trends Biochem. Sci. 2006, 31,
150–155.
(7) Khurana, R.; Ionescu-Zanetti, C.; Pope, M.; Li, J.; Nelson, M.;
Ramírez- Alvarado, L.; Regan, L.; Fink, A. L.; Carter, S. A. Biophys. J.
2003, 85, 1135–1144.
(8) Chiti, F.; Stefani, M.; Taddei, N.; Ramponi, G.; Dobson, C. M.
Nature 2003, 424, 805–808.
(9) Williams, A. D.; Portelius, E.; Kheterpal, I.; Guo, J. T.; Cook, K. D.;
Xu, Y.; Wetzel, R. J. Mol. Biol. 2004, 335, 833–842.
(10) Hortscansky, P.; Christopeit, T.; Schroeckh, V.; Fändrich, M.
Protein Sci. 2005, 14, 2915–2918.
(11) Lashuel, H. A.; LaBrenz, S. R.; Woo, L.; Serpell, L. C.; Kelly,
J. W. J. Am. Chem. Soc. 2000, 122, 5262–5277.
(12) Uversky, V. N.; Segel, D. J.; Doniach, S.; Fink, A. L. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 5480–5483.
(13) Gosal, W. S.; Clark, A. H.; Ross-Murphy, S. B. Biomacromolecules
2004, 2, 2048–2419.
(14) Sipe, J. D. Annu. ReV. Biochem. 1992, 61, 947–975.
(15) Manuelidis, L.; Fritch, W.; Xi, Y.-G. Science 1997, 277, 94–98.
(16) Krebs, M. R. H.; Bromley, E. H. C.; Rogers, S. S.; Donald, A. M.
Biophys. J. 2005, 88, 2013–2021.
(17) Ban, T.; Morigaki, K.; Yagi, H.; Kawasaki, T.; Kobayashi, A.;
Yuba, S.; Naiki, H.; Goto, Y. J. Biol. Chem. 2006, 281, 33677–33683.
(18) Heijna, M. C. R.; Telen, M. J.; van Enckevort, W. J. P.; Vlieg, E.
J. Phys. Chem. B 2007, 111, 1567–1573.
(19) Gosal, W. S.; Ross-Murphy, S. B. Curr. Opin. Colloid Interface
Sci. 2000, 5, 188–194.
(20) Bromley, E. H. C.; Krebs, M. R. H.; Donald, A. M. Faraday
Discuss. 2005, 128, 13–27.
(21) Bassett, D. C. J. Macromol. Sci. Phys. 2003, 42, 227–256.
(22) Magill, J. H. J. Mater. Sci. 2001, 36, 3143–3164.
(23) Murray, S. B.; Neville, A. C. Int. J. Biol. Macromol. 1997, 20,
123–130.
(24) Murray, S. B.; Neville, A. C. Int. J. Biol. Macromol. 1998, 22,
137–144.
(25) Rill, R. L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 342–346.
(26) Fezoui, Y.; Hartley, D. M.; Walsh, D. M.; Selkoe, D. J.; Osterhout,
J. J.; Teplow, D. B. Nat. Struct. Biol. 2000, 7, 1095–1099.
(27) Aggeli, A.; Bell, M.; Carric, L. M.; Fishwick, C. W. G.; Harding,
R.; Mawer, P. J.; Radford, S. E.; Strong, A. E.; Boden, N. J. Am. Chem.
Soc. 2003, 125, 9619–9628.
(28) Hamodrakas, S. J.; Hoenger, A.; Iconomidou, V. A. J. Struct. Biol.
2004, 145, 226–235.
(29) Lockwood, N. A.; van Tankeren, R.; Mayo, K. H. Biomacromolecules
2002, 3, 1225–1232.
(30) Westlind-Danielsson, A.; Arneup, G. Biochemistry 2001, 40, 14736–
14743.
(31) Domike, K. R.; Donald, A. M. Biomacromolecules 2007, 8, 3930–
3937.
(32) Castelletto, V.; Hamley, I. W. Biomacromolecules 2007, 8, 77–
83.
(33) Ruth, L.; Eisenberg, D.; Neufeld, E. F. Acta Crystallogr., Sect. D
2000, 56, 524–528.
(34) Krebs, M. R. H.; MacPhee, C. E.; Miller, A. F.; Dunlop, I. E.;
Dobson, C. M.; Donald, A. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
14420–14424.
(35) Jin, L.-W.; Claborn, K. A.; Kurimoto, M.; Geday, M. A.; Maezawa,
I.; Sohraby, F.; Estrada, M.; Kaminsky, W.; Kahr, B. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 15294–15298.
(36) Taniyama, H.; Kitamura, A.; Kagawa, Y.; Hirayama, K.; yoshino,
T.; Kamiya, S. Vet. Pathol. 2000, 37, 104–107.
(37) Acebo, E.; Mayorga, M.; Val-Bernal, J. F. Pathology 1999, 31,
8–11.
(38) Clark, A. H. G.; Kavanagh, G. M.; Ross-Murphy, S. B. Food
Hydrocolloids 2001, 15, 383–400.
(39) de la Fuente, M. A.; Singh, H.; Hemar, Y. Trends Food Sci. Technol.
2002, 13, 262–274.
(40) Zhang, S. Nat. Biotechnol. 2003, 21, 1171–1178.
(41) Taboada, P.; Barbosa, S.; Castro, E.; Mosquera, V. J. Phys. Chem.
B 2006, 110, 20733–20736.
(42) Juárez, J.; Taboada, P.; Mosquera, V. Biophys. J. 2009, 96, 2353–
2370.
(43) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein
Sci. 1995, 4, 2411–2423.
(44) Bolder, S. G.; Hendrickx, H.; Sagis, L. M. C.; van der Linden, E.
J. Agric. Food Chem. 2006, 54, 4229–4234.
(45) Juárez, J.; Taboada, P.; Mosquera, V. J. Phys. Chem. B 2009, 113,
10521–10529.
(46) Cerda-Costa, N.; Esteras-Chopo, A.; Aviles, F. X.; Serrano, L.;
Villegas, V. J. Mol. Biol. 2007, 366, 1351–1363.
(47) Plakoutsi, G.; Bemporad, F.; Calamai, M.; Taddei, N.; Dobson,
C. M.; Chiti, F. J. Mol. Biol. 2005, 351, 910–922.
(48) LeVine, H., III. Protein Sci. 1993, 2, 404–410.
(49) Krebs, M. R. H.; Bromley, E. H. C.; Donald, A. M. J. Struct. Biol.
2004, 149, 30–37.
(50) Tobitani, A.; Ross-Murphy, S. B. Macromolecules 1997, 30, 4845–
4854.
(51) Ikeda, S.; Nishinari, K. Food Hydrocolloids 2001, 15, 401–406.
(52) Surel, O.; Famelart, M. H. J. Dairy Res. 2003, 70, 253–256.
(53) Saguer, F.; Fort, N.; Alvarez, P. A.; Sedman, J.; Ismail, A. A. Food
Hydrocolloids 2008, 22, 459–467.
(54) Zhao, J.; Majumdar, B.; Schulz, M. F.; Bates, F. S.; Almdal, K.;
Mortensen, K.; Hadjuk, D. A.; Gruner, S. M. Macromolecules 1996, 29,
1204–1215.
(55) Radford, S. E.; Gosal, W. S.; Platt, G. W. Biochim. Biophys. Acta
2005, 1753, 51–63.
(56) Meehan, S.; Berry, Y.; Luisa, B.; Dobson, C. M.; Carver, J. A.;
MacPhee, C. E. J. Biol. Chem. 2004, 279, 3413–3419.
(57) Holm, N. K.; Jespersen, S. K.; Thomassen, L. V.; Wolff, T. Y.;
Sehgal, P.; Thomsen, L. A.; Christiansen, G.; Andersen, C. B.; Knudsen,
A. D.; Otzen, D. E. Biochim. Biophys. Acta 2007, 1774, 1128–1138.
(58) Ikeda, S.; Morris, V. J. Biomacromolecules. 2002, 3, 382–389.
(59) Calamai, M.; Canale, C.; Relini, A.; Stefani, M.; Chiti, F.; Dobson,
C. M. J. Mol. Biol. 2005, 346, 603–616.
182

Additional Supra-Self-Assembly of HSA J. Phys. Chem. B, Vol. 113, No. 36, 2009 12399
(60) Le Bon, C.; Nicolai, T.; Durand, D. Macromolecules 1999, 32,
6120–6127.
(61) Krebs, M. R. H.; Devlin, G. L.; Donald, A. M. Biophys. J. 2007,
92, 1336–1342.
(62) Susi, H.; Byler, D. M. Biochem. Biophys. Res. Commun. 1983, 115,
391–397.
(63) Lin, S.-Y.; Wei, Y.-S.; Li, M.-J.; Wang, S.-L. Eur. J. Pharm.
Biopharm. 2004, 57, 457–464.
(64) Casal, H. L.; Kohler, U.; Mantsch, H. H. Biochim. Biophys. Acta
1988, 957, 11–20.
(65) Fabian, H.; Choo, L.-P; Szendrei, G. I.; Jackson, M.; Halliday,
W. C.; Otvos, L., Jr.; Mantsch, H. H. Appl. Spectrosc. 1993, 47, 1513–
1518.
(66) Lin, S.-Y.; Wei, Y.-S.; Li, M.-J.; Wang, S.-L. Spectrochim. Acta,
Part A 2004, 60, 3107–3111.
(67) Ikeda, S.; Li-Chan, E. C. Y. Food Hydrocolloids 2004, 18, 489–
498.
(68) Ikeda, S.; Nishinari, K. Biopolymers 2001, 59, 87–102.
JP904167E
183

5
10
15
20
25
30
35
40
45
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
Dynamic Article Links ►
PAPER
Hydration effects on the fibrillation process of a globular protein: The
case of human serum albumin.
Josué Juárez, Manuel Alatorre-Meda, Adriana Cambón, Antonio Topete, Silvia Barbosa, Pablo
Taboada*, and Víctor Mosquera.
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
In this work, we have studied the fibrillation process of human serum albumin (HSA) under different
solution conditions. In particular, aggregation kinetics, fibril morphology, and composition structural
changes were investigated at varying experimental conditions such as pH (2.0 and 7.4), temperature (at 25
and 65 ºC), and solvent polarity (ethanol/water mixtures, 10 – 90% v/v). The characterization was carried
out by means of static and dynamic light scattering (SLS and DLS), ThT fluorescence, circular dichroism
(CD) and Fourier Transform Infrared spectroscopies (FT-IR), and transmission electron microscopy
(TEM). The aggregation process and the α-helix to β-sheet transitions were found to be favored by
temperature and physiological pH. Also, pH was observed to influence both the fibrillation pathway and
aggregation kinetics, changing from a classical fibrillation process with a lag phase under acidic
conditions to a downhill polymerization process at physiological pH in the presence of the alcohol.
Regarding protein structural composition, at room temperature and physiological pH ethanol was found to
promote an α-helix to β-sheet conformational transition at intermediate alcohol concentrations, whereas at
low and high ethanol contents α-helix prevailed as the predominant structure. Under acidic conditions,
ethanol promotes an important fibrillation at high cosolvent concentrations due to screening of electric
charges and a decrease in solvent polarity. On the other hand, important differences in the morphology of
the resulting fibrils and aggregates are observed depending on the solution conditions. In particular, the
formation of classical amyloid-like fibrils at physiological pH and high temperature are observed, in
contrast to the usual curly morphology displayed by HSA fibrils under most of solution conditions.
Although high temperature and pH are the main parameters influencing the protein structure
destabilization and subsequent aggregation upon incubation, ethanol helps to regulate the hydrogen
bonding, the attractive hydrophobic interactions, and the protein accessible surface area, thus, modifying
packing constraints and the resulting aggregate morphologies.
Introduction
From a general perspective, amyloid fibril formation present
pros and cons regarding different approaches such as human
health or technological development. On one hand, amyloid
fibrils are recognized as high-performance protein nanomaterials
with a formidable rigidity;
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 1
1-2 on the other, amyloidosis, the
clinical condition in which amyloid fibrils form from innocuous
soluble proteins, has been found to be involved in diverse
pathological responses as observed in various neurodegenerative
disorders including Alzheimer’s, Parkinson’s, and prion diseases
amongst others. 3-5 applications in the area of nanobiotechnology.
Concerning protein folding and its repercussion on amyloid
fibril formation, it has been demonstrated that fibrillation may
proceed from different protein conformations including
completely/partially unfolded
The elucidation of the underlying molecular
mechanisms for fibril formation would result not only in the
development of strategies for the treatment of amyloidosis-related
disorders but also in the design of new biomaterials for future
Grupo de Física de Coloides y Polímeros, Departamento de Física de la
Materia Condensada, Facultad de Física, Universidad de Santiago de
Compostela, E-15782. Santiago de Compostela. Spain.
pablo.taboada@usc.es
6-11 or fully folded states, 12,13
55
although the essential nature of the resulting amyloid fibrils can
be largely independent of the conformational properties of the
soluble precursors. 6
In living organisms, the native environment of proteins is a
60 complex composition of water, cosolvents and cosolutes which
affect the stability of the native protein fold. 14 In this way,
different experimental approaches using the addition of
cosolvents and cosolutes to mimic various cellular environments
in vitro have provided further knowledge on protein folding and
aggregation/fibrillation mechanisms. 15-20 65
Thereby, cosolventmediated
systems have revealed that differences in solvent
185
50

properties induce solvent-adapted structural “responses” of the
aggregating protein, which leads to a structural diversity of the
resulting aggregates/fibers. 21-23 In particular, alcohols are
included among the most commonly studied cosolvents. Several
studies have shown that the effects of alcohol on very different
systems and processes such as the thermal denaturation of nucleic
acids and proteins, protein folding, micellization of surfactant
molecules, or the solubility of nonelectrolytes are strikingly
similar. 24-26 Also, water/alcohol mixtures at moderately low pH
values have been suggested as model systems for studying the
joint action of the local decrease in both pH and dielectric
constant on the protein structure near the membrane surface; 27-32
similarly, new ways of protein administration and delivery based
on inhalation systems with non-aqueous solvents as suspension
media also reinforce the necessity of clarifying how the presence
of the solvent affects the protein conformation and biological
24, 25
activity.
From a thermodynamic standpoint, the protein accessible
surface area (ASA), the solvent exposure of hydrophobic
residues, and a solvophobic backbone 33 can be taken as barriers
hampering protein-solvent interactions. 34 Therefore, the
hierarchical assembly of amyloids can perfectly be understood as
an alternative to the native packing conformational struggle of a
polypeptide chain, reducing ASA and saturating hydrogen
bonding, while a simultaneous decrease in the configurational
entropy of the protein is offset by gains in solvent entropy. The
role of hydrational forces in protein aggreation in vivo, while still
poorly understood, may be instrumental in elucidating the
molecular basis of amyloidosis and, as such, attracts considerable
interest. 35 In this way, the propensity for amyloid formation in
mixed solvents of different proteins as, for example, human
serum albumin (HSA), hen egg white lysozyme, bovine βlactoglobulin,
histone H2A, insulin, and bovine trypsinogen
amongst many others, has been characterized. 36-39 Amongst them,
HSA has been proposed as a good model for protein aggregation
studies 40-42 as a consequence of both its physiological
implications as a carrier protein and blood pressure regulator,
together with its propensity to easily aggregate in vitro. Previous
reports have addressed the HSA fibrillation kinetics, fibrillation
pathway, and intermediate and final protein aggregated structures
under varying pH and ionic strength solution conditions. 40-42
However, a detailed characterization of the aggregation/fibril
process and the structure of the resulting aggregates in mixed
solvent solutions under varying external conditions is still lacked.
In the present work, we present a detailed study on the
aggregation/fibrillation pathway of the protein human serum
albumin (HSA) in water/ethanol mixed solvent solutions at
varying pH and temperature conditions. This enables us to make
a clear comparison between the fibrillation pathway and
intermediate/final protein aggregates in the mixed solvent with
those obtained in pure aqueous solution in order to define the role
of hydrational forces on the protein fibrillation mechanism. In
fact, changes on the fibrillation mechanism from a downhill to a
nucleated-growth polymerization mechanism at acidic pH and
high temperature solution conditions are confirmed. Overall, as a
result of differences in packing depending on the solution
condition, different structures for the resulting fibrils are
observed, from classically thin and very long straight to shorter
5
10
15
20
25
30
35
40
45
50
55
and more curly fibers. In this regard, it comes as no surprise that
factors favoring or disfavoring exposure of solvophobic groups
must affect thermodynamic preferences for particular
conformations.
2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]
186
60
65
Materials and Methods
Materials.
Human serum albumin (70024-90-7), and Thioflavin T (ThT)
were obtained from Sigma Chemical Co. ThT was used as
70 received. All other chemicals were of the highest purity available.
75
80
85
90
95
100
105
110
115
Preparation of HSA solutions.
HSA was purified by liquid chromatography using a
Superdex 75 column equilibrated with 0.01 M phosphate buffer
before use. To prepare the protein solutions, increasing volumes
of ethanol were added to 1.0 mL of HSA stock solution either at
pH 7.4 (sodium monophosphate-sodium diphosphate buffer) or
pH 2.0 (glycine + HCl buffer), to get the desired ethanol
concentration in the mixed solvent. The final protein
concentration in all cases was 10 mg/mL with ionic strength 10
mM. pH was kept constant by adding HCl or NaOH when
needed. The protein concentration was determined
spectrophotometrically, using a molar absorption coefficient of
35219 M -1 cm -1 at 280 nm. 43 Aliquots were added through an
electronic disperser unit Dosimat Metrohm 765. Experiments
were carried out using double distilled, deionized and degassed
water. Before incubation, solutions were filtered through a 0.2
µm filter into sterile test tubes. Samples were incubated at a
specified temperature in a refluxed reactor for 15-20 days as
required.
Light Scattering.
DLS and SLS intensities were measured at 25 °C by means of
an ALV-5000F (ALV-GmbH) instrument working with a
vertically polarized incident light of wavelength λ = 488 nm
supplied by a CW diode-pumped Nd; YAG solid-state laser
supplied by Coherent, Inc. and operated at 400 mW. The intensity
scale was calibrated against scattering from toluene.
Measurements were carried out at a scattering angle θ = 90° to
the incident beam. Solutions were equilibrated for 30 min before
measurements to reach thermal stabilization. Experiment duration
was in the range of 3-5 min, and each experiment was repeated
two or more times. The correlation functions from DLS were
analyzed using the CONTIN method to obtain intensity
distributions of decay rates (Γ). 44 The decay rate distribution
functions gave distributions of apparent diffusion coefficient
(D app = Γ /q 2 ,where the scattering vector q = (4πns/λ)sin(θ /2),
and ns is the refractive index of solvent) and integrating over the
intensity distribution gave the intensity-weighted average of D . app
Values of the apparent hydrodynamic radius (r , radius of
h,app
hydrodynamically equivalent hard sphere corresponding to D ) app
were calculated from the Stokes-Einstein equation:

5
10
15
20
25
r h,app = kT/(6πηD app ) (1)
where k is the Boltzmann constant and η is the viscosity of water
at temperature T.
Thioflavin T spectroscopy.
Protein and ThT were dissolved in buffer at a final proteindye
molar ratio of 50:1. Samples were continuously stirred only
during data acquisition intervals in order to avoid ThT deposition
onto protein aggregates/fibrils. Fluorescence was measured in a
Cary Eclipse fluorescence spectrophotometer equipped with a
temperature controller and a multi-cell sample holder (Varian
Instruments Inc.). Excitation and emission were at wavelengths of
450 and 482 nm, respectively. All intensities were backgroundcorrected
for the ThT-fluorescence in the respective solvent in the
absence and presence of monomeric HSA at pH 7.4 and 2.0.
Circular Dichroism (CD).
Far-UV circular dichroism (CD) spectra were obtained using
a JASCO-715 automatic recording spectropolarimeter with a
JASCO PTC-343 Peltier-type thermostated cell holder. Quartz
cuvettes with 0.2 cm pathlength were used. The protein
concentration was reduced to 0.1 mg/mL in order to avoid light
scattering effects as much as possible due to protein aggregation.
CD spectra were obtained from aliquots withdrawn from the
aggregation mixtures at the indicated conditions after incubation,
and recorded at wavelengths between 195 and 300 nm at 25 ºC.
The mean residue ellipticity θ (deg cm 2 dmol -1 30
) was calculated
35
40
45
50
55
according to: θ=(θObs/10)/(MRM/lc), where θobs is the
observed ellipticity (in deg), MRM is the mean residue molecular
mass (in g mol -1 ), l is the optical path-length (in cm), and c is the
protein concentration (in g ml -1 ). The secondary structure of the
protein was calculated from far-UV CD spectra by running the
SELCON3, CONTIN, and DSST programs within the
DICHROWEB server. 45,46 Final results were assumed when data
generated from all programs showed convergence.
Transmission electron microscopy (TEM).
HSA solutions were applied to carbon-coated copper grids,
blotted, washed, negatively stained with 2% (w/v) of
phosphotungstic acid, air dried, and then examined with a Phillips
CM-12 transmission electron microscope operating at an
accelerating voltage of 120 kV. Samples were diluted between
20-200-fold prior deposition onto the grids.
Results and Discussion
To induce HSA aggregation and subsequent amyloid
formation we use 10 mg/mL HSA solutions at either pH 7.4 or
pH 2.0 and ionic strength of 10 mM. At pH 7.4 the protein is in
its native state whereas at pH 2.0 it is in its E-expanded one,
which is characterized by a separation of domain III and a loss of
the intradomain helices of domain I. 47 Monomer sizes and
structural compositions were similar to those previously
reported. 40 Samples were prepared by adding specified volumes
of protein stock solutions and different amounts of ethanol, which
has been shown to destabilize both the secondary and tertiary
structures of HSA; 36 subsequently, samples were incubated at
room or elevated temperature (65 ºC, above the melting
temperature of HSA) 48,49 to induce further protein structural
changes and aggregation.
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 3
187
60
65
70
75
80
85
90
95
100
105
110
Aggregate formation.
The formation of HSA aggregates and their aggregation
kinetics were firstly analyzed by SLS. Figure 1 shows the time
evolution of the scattered light intensity of HSA samples at
varying pH, temperature and alcohol content conditions upon
incubation. Different curve profiles are obtained depending on
incubation conditions.
At physiological pH (Figure 1a-b), the plots reflect a quick
increase in the scattered intensity at short incubation times, which
denotes the fast formation of protein aggregates. SLS plot profiles
suggest a mechanism of continuous protein aggregation (in
particular, a continuous fibrillation process as later confirmed by
ThT, CD and TEM data) without a nucleation step, as denoted the
absence of a lag phase in the early periods of incubation (see inset
in Figure 1a). In addition, the absence of a faster aggregation
process when protein seeds (i.e. protein aggregates) are added to
protein solutions followed by subsequent incubation, and the
decrease in the aggregation rate if the starting protein
concentration is lowered (see Figure S1 in ESI) further indicate
that under the present conditions HSA aggregation occurs by
means of a classical coagulation or downhill polymerization
mechanism, i.e. each protein monomer association is bimolecular,
effectively irreversible and thermodynamically favorable, so that
there is no energy barrier to impede aggregate growth. 50 A similar
behavior has been previously obtained in the absence of
alcohol. 40-42,50,51 ThT fluorescence data confirmed similar trends
as those observed by SLS (see Figure S2). High values of ThT
fluorescence point to an important formation/gain of β-sheet
structure along the protein aggregation process, which is a first
evidence of the possible formation of HSA fibrils during the
aggregation process, 37 as further confirmed below by CD and
TEM techniques.
Also, under physiological conditions we observe that as the
ethanol concentration increases up to 60% (v/v) the formation of
more numerous and/or bulkier protein aggregates takes place as
derived from both the increments in scattered intensities –
proportional to protein concentration and molecular weight
(Figure 1a,b), and the shifts of the aggregate size distributions to
larger values (see Figure 2a-b below). The clustering of alcohol
molecules and the enhancement of alcohol-water interactions at
intermediate ethanol concentrations in the mixed solvent should
modify the extent of protein hydration and molecular packing 53 as
confirmed by CD and FT-IR below, favoring an enhanced protein
aggregation. At larger alcohol concentrations (> 60% (v/v)),
protein solutions became progressively turbid as a consequence
of the formation of even larger aggregates, 52 which gradually
precipitated (see below for further explanation).

5
10
15
20
25
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
Intensity (a.u.)
Intensity (a.u.)
1400
1200
1000
800
600
280
240
200
160
120
80
40
0
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
Time (min)
100
80
60
40
20
0
Intensity (a.u.)
25
20
15
10
5
0
0 500 1000 1500 2000 2500 3000
Time (min)
0 2000 4000 6000
Time (min)
8000 10000 12000
Dynamic Article Links ►
PAPER
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 4
188
a)
c)
Intensity (a.u)
Intensity (a.u.)
1200
1100
1000
900
800
100
50
0
24
20
16
12
8
4
Intensity (a.u.)
30
25
20
15
10
5
0 5 10 15 20 25 30
Time (min)
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
Time (min)
0
0 5000 10000 15000 20000 25000
Time (min)
Figure 1: Time evolution of the scattered intensity of HSA solutions at a) pH 7.4 and 65 ºC, b) pH 7.4 and 25 ºC, c) pH 2.0 and 65 ºC,
and d) pH 2.0 and 25 ºC under different concentrations of ethanol in the mixed solvent. In a), b) and d) ethanol concentrations are ()
20, () 40, () 60, and () 80% (v/v). The enhanced scattered dispersion above 1000 a.u. at high alcohol concentrations in the mixed
solvent is a consequence of the formation of big aggregates. In c) ethanol concentrations in the mixed solvent are () 20, () 40, ()
50, () 60, and () 80% (v/v). The inset in Figure 1b illustrates, as an example, the absence of a lag phase in the incubation of HSA at
25 ºC in the presence of 20% (v/v). The inset in Figure 1c illustrates the existence of the first step in the aggregation kinetics.
For samples incubated under acidic conditions (Figure 1c-d),
the scattered intensity plot profiles depend on both alcohol
content and temperature. HSA samples incubated at 25 ºC in the
whole range of mixed solvent compositions and those incubated
at 65 ºC at ethanol concentrations below 50% (v/v) gave rise to
SLS plots with classical sigmoidal profile, i.e., there exists an
initial lag phase in which the scattered intensity remains almost
constant followed by a subsequent exponential phase which ends
up in a final equilibrium region. These three stages correspond to
the nucleation, elongation, and equilibrium phases. 54,55 The
presence of a lag phase at acidic pH is related to the different
initial structure of protein molecules in acidic solution, and to
changes in the nature and strength of intermolecular interactions
upon incubation, as previously stated in previous works. 41,42 This
finding is additionally confirmed when a seeding growth process
is performed, in which the lag phase is abolished (see Figure
S3a).
On the other hand, for HSA samples at 65 ºC in the presence
of alcohol concentrations between 50 and 90% (v/v), the
formation of protein aggregates occurs in two well-differentiated
30
35
40
45
steps: there exists a first short increase in the scattered intensity at
very low incubation times followed by a quasi-plateau region;
then, a second much stepper rise in a narrow time interval takes
place until the final equilibrium region is reached. It has been
previously demonstrated that this plot profile emerges when small
oligomeric structures rapidly form, 40 which need more time to
develop and/or persist for longer times due to their enhanced
solubility under acidic conditions. 40-41 These species are watersoluble
and, hence, only after achieving a critical
concentration/size the energy landscape of the solution changes,
i.e., the aggregation process becomes thermodynamically
favorable enabling the evolvement of small oligomers to larger
aggregates 56 as confirmed below by DLS data. Hence, the quasiplateau
region has been identified as a lag phase, as confirmed its
suppression when a seeding fibril growth process is performed
under the present solution conditions (see Figure S3b). It is also
worth mentioning that the scattered intensities at equilibrium
increases at ethanol concentrations between 0 and 50% (v/v),
followed by a decrease between 50-70% (v/v), and by a new final
increment (see Figure 1c). As confirmed by TEM and CD data
b)
d)

5
10
15
20
25
30
35
40
45
50
below, the first increase is related to the presence of progressively
interconnected aggregates from the reduction in solvent polarity,
which originates subtle changes in the protein secondary structure
composition favoring the entanglement of protein aggregates. The
subsequent intensity decrease at larger alcohol concentrations (>
50% v/v) originates from the formation of well-dispersed
aggregates (short fibrils, see TEM pictures below) as a
consequence of additional changes in protein secondary structure
(as confirmed CD data below). The final scattered intensity
increase at the largest ethanol contents arises from the presence of
more numerous and longer aggregated structures in solution.
These observations are in contrast with the behavior observed
at 25 ºC (see Figure 1d). For samples containing low ethanol
contents, their low scattering intensity suggests that almost no
aggregation occurs, whereas at higher ethanol contents (>40%
v/v) the rise in the scattered intensity profile corroborates the
presence of aggregates as a consequence of the screening of
electrostatic repulsions between protein molecules as the solvent
permittivity decreases. Additional confirmation is given by CD
and ThT fluorescence data below.
On the other hand, comparison of scattered intensities at
acidic and physiological pH shows the scattered intensity at the
former pH is, in general, lower than that at the latter at the same
mixed solvent conditions (see Figure 1). This is a consequence of
the lowering of intermolecular interactions due to protonation of
positively charged residues. 57 Also, when comparing the effect of
temperature on the scattered intensity profiles, we can observe
that incubation at 25 ºC involves a lower scattered intensity at
both pH in the whole range of solvent compositions. It is known
that hydrogen bonding is weakened as temperature rises, but
hydrophobic interactions become strengthened. 58 As the heating
process proceeds, destabilization of the protein helical structure
of HSA occurs though hydrogen bonding weakening, as denoted
by the loss of the minima at 208 and 222 nm typically assigned to
α-helices in the HSA native state in the CD profiles of HSA
samples at high temperature (see Figure 3); then, previously
buried aminoacid residues can be now exposed to solvent as
follows from changes in protein conformation from CD data (see
below), which results more prone to aggregation as observed
from the SLS data (and further confirmed by TEM pictures
below).
Aggregation kinetics.
Data analysis of scattered intensity plots with a sigmoidal-like
profile is consistent (also according to spectroscopy and TEM
data), at a first approximation, with the following kinetic scheme:
M ↔ I ↔ nucleus → fibrils
where M is the monomer and I is the intermediate. Thus, the
scattered intensity as a function of time was fitted to the
following equation:
(2)
where Y is the scattered intensity, x the time, and xo is the time to
reach 50% of maximal scattered intensity. Thus, the apparent rate
constant, kapp, of fibril growth is given by 1/τ, and the lag time by
xo - 2τ. 59 Lag times and apparent first-order rate constants were
determined from curve fits and shown in Table S1 in ESI. In
general, an increase in the alcohol concentration resulted in
longer lag times and larger apparent rate constants. Also, the
aggregation kinetics at physiological pH appears to be faster than
under acidic conditions. This behavior is probably related to the
existence of electrostatic repulsions between protein molecules
and to an enhanced solubility of the initial protein clusters under
acidic conditions. Finally, it is necessary to mention that two
growth rates were obtained at high ethanol concentrations upon
incubation at acidic conditions and 25 ºC. This fact is originated
by the presence of intermediate oligomeric structures along the
HSA aggregation pathway under these solution conditions, as
discussed elsewhere. 40
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 5
55
60
65
70
75
80
85
90
95
100
105
Aggregate size distribution.
Figure 2 shows some examples of population size
distributions for samples incubated under the different solution
conditions. At 65 ºC and physiological pH, two different
populations are well differentiated and present under all mixed
solvent compositions (Figure 2a): A peak at large sizes (ca. 400-
1000 nm) assigned to protein aggregates, and other at smaller
sizes (at ca. 20 to 40 nm) probably representing protein
clusters/oligomers. Also, it is observed that a third population of
aggregates with very large sizes (∼ 20 µm) appears at an ethanol
concentration of 80% (v/v). This feature confirms that supraaggregation
of protein aggregates is favored at high alcohol
contents under the present conditions, in agreement with SLS
data, and as further confirmed below by TEM pictures. For
samples incubated at 25 ºC and physiological pH and at 65 ºC and
pH 2.0 (Figure 2b-c), a progressive increase in the size of protein
aggregates takes place as the ethanol concentration increases, as
denoted by the existence of bimodal distributions. Only at
intermediate ethanol concentrations the referred size increase
involves the superimposition of population sizes corresponding to
large protein aggregates and oligomeric structures, creating a
wide population distribution (but still bimodal, as observed after
deconvolution). In contrast, incubation at pH 2.0 and 25 ºC
results in the simultaneous decrease of the peak height
corresponding to the smallest sizes and the increase of that
corresponding to the biggest ones. This fact indicates a
progressive evolvement of protein monomers and small
oligomeric structures to aggregates of larger sizes as the ethanol
concentration in the mixed solvent increases (Figure 2d),
corroborating SLS data.
Regarding the hydrodynamic radii derived from the size
distributions depicted in Figure 2a-d, they can be grouped into
three average values corresponding to rh,app of ca. 400-1000 nm,
20-50 nm, and 2-5 nm. At this point, it is necessary to remind that
DLS sizes are obtained assuming a spherical shape, so these
189

5
10
15
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
Intensity (a.u.)
Intensity (a.u.)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1 10 100 1000 10000 100000
r (nm)
h,app
1 10 100 1000 10000 100000
r (nm)
h,app
r h,app (nm)
900
800
700
600
500
400
300
200
100
e)
a)
c)
Dynamic Article Links ►
0.1 1 10 100
r (nm)
h,app
1000 10000 100000
PAPER
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 6
190
Intensity (a.u.)
Intensity (a.u.)
0
10 20 30 40 50 60 70 80 90 100
% (v/v) ethanol
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.1 1 10 100
r (nm)
h,app
1000 10000 100000
Figure 2: Selected size distributions derived from DLS data for HSA solutions incubated at pH 7.4 and a) 65ºC, and b) 25 ºC at ethanol
concentrations of (⎯) 20, (⎯) 60, and (⎯) 80% (v/v), respectively; and at pH 2.0 and c) 65 ºC and d) 25 ºC at ethanol concentrations of
(⎯) 20, (⎯) 50, and (⎯) 80% (v/v), respectively. Figure 2e shows the variation of the hydrodynamic radii of the main peak of the
distribution with ethanol concentration after incubation at 65º C and () pH 7.4 and () 2.0, or at 25 ºC and () pH 7.4 and () 2.0.
are bare size estimations. In general, aggregates formed at
physiological pH are bulkier if compared to those formed under
acidic conditions, as plotted in Figure 2e. Structures with rh,app =
400-1000 nm constitute the main contribution to light scattering.
As depicted in the TEM images shown below, vide infra, these
can be associated with fibrillar conformations, in particular,
assigned to the large axis of protein fibers. Those values of rh,app between 30-50 nm might have different interpretations depending
on solution conditions: On one hand, at acidic pH and 25 ºC they
correspond to oligomeric structures, which confirms the two-step
20
25
aggregation process observed from SLS data under these
conciditions; on the other, at high temperature conditions rh,app values might also include contributions from the fibrillar
aggregates width. Finally, the smallest rh,app values around 2-5 nm
fit fairly well with the estimated size of protein monomers.
Conformational structure changes along protein fibrillation.
To get further insight into the structural characterization of the
resulting protein aggregates, CD, FT-IR and ThT fluorescence
d)
b)

5
10
15
20
25
30
55
Figure 3: Selected far-UV spectra of HSA solutions after incubation at pH 7.4 at a) 65 ºC and b) 25 ºC; and at pH 2.0 at c) 65 ºC and d)
25 ºC at different ethanol concentrations in the mixed solvent: 1) 20, 2) 40, 3) 60, and 4) 80% (v/v).
experiments were performed. Figure 3 represents the far-UV CD
spectra of HSA solutions at the different experimental conditions.
When incubation is performed at high temperature and
physiological pH (Figure 3a), a minimum at ca. 215 nm is
observed in the whole range of solvent mixture compositions.
This minimum denotes the predominant presence of β-sheet
structure. The amount of α-helix conformation remains constant
up to a 50% (v/v) alcohol concentration (see Table S2) and, then,
it slightly decreases with an additional slight gain of β-sheets. At
the largest ethanol concentrations (> 80% v/v), the reduction
observed in ellipticity is related to light scattering caused by the
presence of big aggregates in solution. These aggregates do not
enable the obtention of reliable structural composition values.
Upon incubation at pH 7.4 and 25 ºC (Figure 3b), a little increase
in α-helical content is observed at ethanol concentrations below
30% (v/v) as a result of the weakening of hydrophobic
interactions due to the presence of the cosolvent. 36 This fact
results in the stabilization of the compact protein native structure
and strengthens other interactions such as hydrogen bonding,
stabilizing the secondary structure. Thus, alcohol acts as a
“structure maker”. At larger ethanol concentrations (30-60 %
(v/v)) a simultaneous decrease in α-helix and an increase in β-
sheet structures take place in a wider alcohol range as compared
to non-buffered solutions 36 , which is characterized by the absence
of the minimum at 220-224 nm and the appearance of a new one
at ca. 215 nm 60,61 (see Figure 3b). At ethanol contents higher than
60% (v/v), a progressive new increase in ellipticity at 222 nm and
208 nm occurs, which suggests the formation of non-native
helical structure at the expense of β-sheet and unordered
conformations. The loss of non-polar contacts at high ethanol
concentrations appears to favor the formation of intramolecular
hydrogen bonds responsible of the formation of new helices. This
non-native α-helix structure is more prone to aggregation, 62 as
denoted by the important increase in aggregate sizes previously
observed in Figure 2.
Incubation at acidic pH and high temperature (Figure 3c) was
found to involve an increasingly negative Cotton effect at 208 nm
and the simultaneous decay in ellipticity at 222 nm. This suggests
that, while ethanol induces disorder, some helical content is still
retained. In fact, within the ethanol concentration range between
20-50% (v/v), the α-helix and unordered conformation contents
slightly increase at the expense of β-sheets (see Table S2). The
formation of this extra α-helix might arise from the disruption of
long-range hydrophobic interactions due to the presence of
alcohol molecules, which allows local helical conformation to
provide a transient means of decreasing backbone solvation. At
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 7
35
40
45
50
191

5
10
15
20
25
Figure 4: Normalized maximum ThT fluorescence intensities of HSA samples incubated at different ethanol concentrations in the mixed
solvent at a) pH 7.4 and 65 ºC, b) pH 7.4 and 25 ºC, c) pH 2.0 and 65 ºC and d) pH 2.0 and 25 ºC. Normalization is made with respect to
the maximum ThT fluorescence observed at pH 7.4 and 65 ºC.
30 ethanol concentrations larger than 50% (v/v) the β-sheet content
increases, as denoted from the progressive shift of the minimum
in CD spectra to 215 nm, which is characteristic of β-strand
formation. In contrast, at acidic pH and 25 ºC (Figure 3d), the
structural content of protein samples at ethanol concentrations
35 lower than 60% (v/v) remains almost invariable. Since HSA
molecules are in a starting acid-denaturated state, alcohol
stabilizes the α-helix conformation of the acid-unfolded protein
monomers by minimizing the exposure of the peptide backbone.
In particular, at alcohol concentrations < 40% (v/v),
40 intermolecular interactions are disfavored by suppression of the
strong aggregate-stabilizing effect of negatively charged residues,
still resulting in an effective electrostatic repulsion between
positively charged chains. Hence, under these conditions alcohol
stabilizes the molten-globule state of HSA during incubation, and
only protein clusters can be formed. 63,64 45
The presence of these
clusters, confirmed by the presence of a small peak at relatively
low aggregate sizes (∼20-30 nm) (see Figures 2d), implies some
increase in light scattering from solution as shown previously
and, thus, possibly originates the slight decrease in ellipticity
50 observed in Figure 3d. Protein molecules experience additional
structural rearrangements at ethanol concentrations between 50-
90% (v/v) when the polarity of the medium is drastically
changed. This involves a decrease in α-helix structure and
formation of β-strands that, as observed from light scattering
55 data, are prone to aggregate. FT-IR measurements corroborate the
structural changes undergone by protein molecules depicted by
CD data (for additional comments see ESI).
Although reliable in detecting β-strands and probing hydrogen
bonding between them, CD and FT-IR spectroscopies fail to
60 discriminate between amorphous and ordered aggregates. Since
ThT dye strongly emits when bound to amyloid-like material
rather than to amorphous aggregates, we conducted ThT
fluorescence measurements of HSA samples under the different
solution conditions in order to determine the presence of ordered
65 aggregates (amyloid-like fibrils) in solution. Experimental data
were background corrected for solvent and monomeric protein
contributions and normalized to the largest value attained, i.e., at
pH 7.4 and 65 ºC at 70 % (v/v) ethanol. Figure 4 confirms that βsheet
rich structures (fibrils as shown by TEM pictures below) are
70 formed under all conditions except at acidic pH and 25 ºC at
alcohol concentrations below 60% (v/v). At pH 7.4 and 65 ºC
(Figure 4a), the amount of fibrils progressively increases up to an
ethanol content of 70% (v/v), and then decreases. This decrease
in ThT fluorescence can be associated with the presence of
75 mature fibril association/aggregation, as confirmed by TEM (see
below), which may reduce the protein exposed surface area and
the number of available ThT binding sites. 65 In contrast, at acidic
conditions (Figure 4c) the maximum level of fibril formation
takes place at the largest ethanol concentrations (70-90% v/v). On
80 the other hand, the temporal evolution of ThT binding confirms
8 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]
192

10
15
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
a)
c) d)
e) f)
Dynamic Article Links ►
PAPER
Figure 5: Selected TEM images of HSA samples incubated at pH 7.4 and a-c) 65 ºC, or d-f) 25 ºC at ethanol concentrations in the mixed
solvent of a,d) 20, b,e) 60, c,f) 80% (v/v), respectively.
the trends previously observed from SLS kinetic data, although
some differences at acidic and high temperature conditions are
observed (see Figure S2 and further explanation in ESI).
Based on all the above findings, the combination of i)
incubation at high temperature and ii) the presence of ethanol as a
cosolvent in solution result in important variations of the protein
structural content and subsequent strengthening of hydrophobic
interactions between protein molecules. Incubation at high
temperatures involves the denaturation of protein monomers due
to the disruption of the structure of protein domains 48,49,63 while
the presence of the cosolvent changes the solvent polarity and
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 9
193
5
20
25
30
b)
induces a further exposure of residues, 66 which enhances the
internal degrees of freedom of side-chains. 67 Then, in most of the
studied conditions, the cosolvent induces additional modifications
in the starting protein structure, leading to a strong protein
aggregation and fibril formation, but with differences in the
packing mechanisms leading to fibril polymorphism, as will be
shown below. All these facts become even clearer when
comparing the secondary structure compositions here obtained
with those measured under identical incubation conditions in the
absence of alcohol. 36,40-42

Aggregate morphology.
In order to determine the structural arrangement of protein
aggregates and confirm the presence of amyloid-like fibrils, TEM
5 images were obtained. Figures 5 and 6 show the structures of the
different protein aggregates upon incubation under the different
mixed solvent conditions.
Samples incubated at 65 ºC and physiological pH fibrillated
in the whole range of alcohol concentrations analyzed (Figure 5a-
10 c). However, remarkable differences in the structure of the
resulting fibrils are observed if compared to those obtained in the
absence of ethanol. In particular, for alcohol concentrations up to
70% (v/v) straight-several micrometers-long fibers with widths of
ca. 10-12 nm are seen (Figure 5a,b). These fibrils are longer than
15 those obtained in the absence of ethanol under similar conditions
and do not possess the typically observed curly morphology of
HSA fibrils; 40,68 In fact, they resemble those fibers obtained for
classically amyloidogenic proteins such as α-synuclein, 69
insulin, 67 or lysozyme. 70 Probably, the larger β-sheet content
20 which results from the combination of high temperature and
alcohol-mixed solvent conditions during incubation favored the
establishment of strong attractive interactions between protein
monomers and a further exposition of their side chains to solvent.
The reduction of the thermodynamic costs for the exposure of
25 hydrophobic residues as the dielectric properties of the solvent
approach to those of the bulk alcohol may be the origin for such
behavior. Hence, this enables the possibility of a better packing of
the protein strands inside the fibrils favoring an increase in fibril
length. At larger ethanol contents (> 70% v/v), an important
30 amount of thicker mature fibrils formed by lateral assembly (side
by side) of two or more individual fibrils is observed. Moreover,
very large aggregates composed of assembled fibrils are also seen
(Figure 5c), which agrees with the decrease in ThT fluorescence
and the existence of a third population distribution at very large
35 sizes observed by DLS.
As observed from Figure 5d-f, incubation at room
temperature and physiological pH involved the formation of
protein globules (with sizes ranging from 15 to 60 nm), which
seem to elongate at low alcohol content (< 30% v/v) as a
40 consequence of the existence of attractive interactions between
them (see Figure 5d). As ethanol concentration increases (up to
60% v/v) we observe the formation of curly fibrils with similar
characteristic features (lengths between 0.2-1 µm, widths ranging
between 8-15 nm, and a curly morphology) as those obtained
45 upon incubation at high temperature in the absence of the alcohol
(Figure 5e). 40 At larger ethanol contents (>60% v/v) fibrils
progressively disappeared and large-elongated glomerular
aggregates were observed. Considering CD, FTIR, and ThT
fluorescence data obtained at this alcohol concentration range, we
50 expect these aggregates to possess an increased level of α-helix
content (Figure 5f) and recover the tertiary conformation. 36
Under acidic conditions and 65 ºC, more curly fibrils are
observed if compared with incubation at 25 ºC (see Figure 6a and
d, for example). Also, the width and length of these fibrils are
55 slightly larger than at room temperature. At intermediate ethanol
contents, interconnected networks of elongated aggregates are
observed (Figure 6b), which agrees with the increases in scattered
light observed by SLS data. At large ethanol concentrations (>
60% v/v), single dispersed curly fibrils are again observed
60 (Figure 6c), in agreement with the decrease in SLS intensities and
conformational CD and FTIR data. On the other hand, at acidic
pH and 25 ºC oligomeric partially-elongated structures with
lengths of ca. 20-40 nm and widths of 3-4 nm are observed at
ethanol concentrations between 0-40% (v/v), as seen in the
absence of alcohol (see Figure 6d). 40 According to ThT and CD
data, these structures are mainly composed of α-helix and
unordered conformations. When larger amounts of ethanol are
present in the solvent (>60% v/v), fibrils with a curly morphology
are present (see Figures 6e,f). Under the present conditions, it
seems the increasing amounts of ethanol induce an enlargement
and fusion of oligomers as a result of additional intermolecular
hydrophobic contacts between side chains, which favors the
formation of β-strands and, hence, fibril formation.
10 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]
65
70
75
80
85
90
95
100
105
110
115
Conclusions
In summary, from a general perspective, ordered
aggregation of globular proteins requires partial unfolding of the
native state. 71 This enables the globular precursor state to be
populated and to expose aggregation-competent regions that are
usually protected against intermolecular interactions in the native
protein. However, these regions can be different and display
distinct propensities to solvent exposition depending on solution
conditions and, thus, different ability to fibrillate. 40,72 This seems
to be the origin of the differences observed in the behavior of
HSA samples incubated at physiological and acidic pH at the
different solvent compositions. Under physiological pH ethanol is
able to destabilize the protein secondary structure in most of the
conditions, giving rise to an aggregation/fibrillation process. In
contrast, under acidic conditions pH induces further structural
changes in secondary structure favoring electrostatic repulsive
interactions, which makes the protein molecules more soluble,
disfavoring their aggregation to a great extent. Only when a
cosolvent concentration threshold is reached does the fibrillation
process start to be significant. Despite amyloid formation can
occur under different solution conditions, rates and extents of
fibril formation, fibrillation pathways, and the structures of
resulting fibrils can be very diverse depending on solution
conditions. Constraints due to electrostatic and hydrophobic
interactions are, in this regard, important in that they govern the
side-chain packing that controls stacking of β-sheets, favoring the
formation of more classical amyloid-like fibrils at physiological
pH and high temperature. The different modes of association
under the present conditions are also dictated by the influence of
solvent on hydrogen bonding within the β-sheets. At this respect,
the distinct self-assembled motifs can be also a result of changes
in the arrangement of β-strands within the β-sheets. This is clear
from the different types of fibrillar structures observed under
varying ethanol concentrations, in particular at physiological pH:
from the classical curly geometry of HSA fibrils at low ethanol
concentrations to the formation of long-straight fibrils which
resemble those formed in originally disease-related proteins.
Nevertheless, high temperature and physiological pH conditions
are the main parameters influencing the protein structure
destabilization and subsequent aggregation upon incubation,
while ethanol aids to regulate hydrogen bonding, the attractive
hydrophobic interactions, and the protein accessible surface area.
194

5
10
15
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
a)
c) d)
e) f)
Dynamic Article Links ►
PAPER
Figure 6: Selected TEM images of HSA samples incubated at pH 2.0 and a-c) 65 ºC, or d-f) at 25 ºC at ethanol concentrations in the
mixed solvent of a,d) 20, b,e) 50, c,f) 80% (v/v), respectively.
The role of “fibril enhancer” of ethanol under most of the
conditions is contrary to what is observed when already-formed
fibrils are incubated in its presence. For example, it has been
shown that insulin and β-lactoglobulin fibrils disassemble upon
incubation in DMSO and ethanol, respectively, as a result of a
weakening of hydrogen bonding inside the fibrils. 73,74 consequence of changes in the energy landscape of the
aggregation process.
20 Acknowledgement. Authors thank Ministerio de Ciencia e Innovación
(MICINN) for research project MAT 2010-17336, Xunta de Galicia for
research project INCITE09206020PR and for European Regional
Thus, it Development Funds (research project 2010/50), and Fundación Ramón
appears that the solvent composition and the different initial state Areces for additional financial support.
of the protein are responsible of the observed differences as a 25
Electronic Supplementary Information (ESI) available:
Complementary seeding growth and concentration-dependent kinetic
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 11
195
b)

5
10
15
20
25
30
35
40
45
50
55
60
65
studies, tables containing kinetic constants derived from SLS experiments
and secondary structure compositions under the different solution
conditions, FTIR spectra and discussion, and temporal evolution of ThT
biding with subsequent discussion. See DOI: 10.1039/b000000x/
References
1. T. P. Knowles, A. W. Fitzpatrick, S. Meehan, H. R. Mott, M.
Vendruscolo, C. M. Dobson, M. E. Welland, Science 2007,
318, 1900-1903.
2. S. G. Zhang, Nature Biotechnol. 2003, 21, 1171-1178.
3. A. T. Alexandrescu, Protein Sci. 2005, 14, 1-12.
4. K. Lundmark, G. T. Westermark, A. Olsen, P. Westermark,
Proc. Natl. Acad. Sci. USA 2005, 102, 6098-6102.
5. M. B. Pepys, Ann. Rev. Med. 2006, 57, 223-241.
6. M. Calamai, F. Chiti, C. M. Dobson, Biophys. J. 2005, 89,
4201-4210.
7. B. A. Vernaglia, J. Huang, E. D. Clark, Biomacromolecules
2004, 5, 1362-1370.
8. S. Goda, K. Takano, K. Yutani, Y. Yamagata, R. Nagata, H.
Akutsu, S. Maki, K. Namba, Protein Sci. 2000, 9, 369-375.
9. F. G. de Felice, M. N. N. Vieira, M. N. L. Meirelles, L. A.
Morozova-Roche, C. M. Dobson, S. T. Ferreira, FASEB J.
2004, 18, 1099-1101.
10. A. E. Cao, D. Y. Hu, L. H. Lai, Protein Sci. 2004, 13, 319-324.
11. M. R. H. Krebs, D. K. Wilkins, E. W. Chung, M. C.
Pitkeathly, A. K. Chamberlain, J. Zurdo, C. V. Robinson, C.
M. Dobson, J. Mol. Biol. 2000, 300, 541-549.
12. F. Bemporad, G. Calloni, S. Campioni, G. Plakoutsi, N.
Taddei, F. Chiti, Acc. Chem. Res. 2006, 39, 620-627.
13. G. Soldi, F. Bemporad, S. Torrassa, A. Relini, M. Ramazzotti,
N. Taddei, F. Chiti, Biophys. J. 2005, 89, 4234-4244.
14. S. N. Timasheff, Ann. Rev. Biophys. Biomol. Struct. 1993, 22,
67-97.
15. W. Dzwolak, Biochim. Biophys. Acta 2006, 1764, 470-480.
16. N. Rezaei-Ghaleh, M. Zweckstetter, D. Morshedi, A. Ebrahim-
Habibi, M. Nemat-Gorgani, Biopolymers 2009, 91, 28-36.
17. J. H. Lee, G. Bhak, S. G. Lee, S. R. Paik, Biophys. J. 2008, 95,
L16-L18.
18. M. Holley, C. Eginton, D. Schaefer, L. R. Brown, Biochem.
Biophys. Res. Comm. 2008, 373, 164-168.
19. V. Castelletto, I. W. Hamley, P. J. F. Harris, U. Olsson, N. J.
Spencer, Phys. Chem. B 2009, 113, 9978-9987.
20. I. W. Hamley, D. R. Nutt, G. D. Brown, J. F. Miravet, B.
Escuder, F. Rodríguez-Llansola, J. Phys. Chem. B 2009, 114,
940-951.
21. A. K. Paravastu, A. T. Petkova, R. Tycko, Biophys. J. 2006,
90, 4618-4629.
22. W. Hoyer, T. Antony, D. Cherny, G. Heim, T. M. Jovin, V.
Subramaniam, J. Mol. Biol. 2002, 322, 383-393.
23. D. P. Smith, S. Jones, L. C. Serpell, M. Sunde, S. E. Radford,
J. Mol. Biol. 2003, 330, 943-954.
24. J. S. Patton, R. M. Platz, Adv. Drug Delivery Rev.1992, 8, 179-
196.
25. W. S. Choi, G. G. K. Murthy, D. A. Edwards, R. Langer, A.
M. Klibanov, Proc. Natl. Acad. Sci. USA 2001, 98, 11103-
11107.
26. S. Cinelli, G. Onori, A. Santucci, Colloids Surfaces A 1999,
160, 3-8.
27. V. E. Bychkova, O. B. Ptitsyn, Biochem. Mol. Biol. 1993, 4,
133-163
28. O. B. Ptitsyn, V. E. Bychkova, V. N. Uversky, Philos. Trans.
Royal Soc. London B-Biol. Sci. 1995, 348, 35-41.
29. V. E. Bychkova, A. E. Dujsekina, S. I. Klenin, E. E. Tiktopulo,
V. N. Uversky, O. B. Ptitsyn, Biochemistry 1996, 35, 19,
6058-6063.
30. V. N. Uversky, N. V. Narizhneva, S. O. Kirschstein, S. Winter,
G. Löber, Folding Design 1997, 2, 163-172.
31. Y. O. Kamatari, T. Konno, M. Kataoka, K. Akasaka, J. Mol.
70 Biol. 1996, 259, 512-523.
32. N. V. Narizhneva, V. N. Uversky, Protein Pept. Lett. 1997, 4,
243-249.
33. Y. X. Qu, C. L. Bolen, D. W. Bolen, Proc. Natl. Acad. Sci.
USA 1998, 95, 9268-9273.
34. S. N. Timasheff, Proc. Natl. Acad. Sci. USA 2002, 99, 9721-
12 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]
75
80
85
90
95
100
105
110
115
120
125
130
135
9726.
35. D. Liu, T. Wyttenbach, C. J. Carpenter, M. T. Bowers, J. Am.
Chem. Soc. 2004, 126, 3261-3270.
36. P. Taboada, S. Barbosa, E. Castro, M. Gutiérrez-Pichel, V.
Mosquera, Chem. Phys. 2007, 340, 59-68.
37. P. Taboada, S. Barbosa, E. Castro, V. Mosquera, J. Phys.
Chem. B 2006, 110, 20733-20736.
38. Y. Aso, K. Shiraki, M. Takagi, Biosci. Biotech. Biochem.
2007, 71, 1313-1321.
39. W. Dzwolak, S. Grudzielanek, V. Smirnovas, R. Ravindra, C.
Nicolini, R. Jansen, A. Loksztejn, S. Porowski, R. Winter,
Biochemistry 2005, 44, 8948-8958.
40. J. Juárez, P. Taboada, V. Mosquera, Biophys. J. 2009, 96,
2353-2370.
41. J. Juárez, S. Goy-López, A. Cambón, P. Taboada, V.
Mosquera, J. Phys. Chem. B 2009, 113, 10521-10529.
42. J. Juárez, P. Taboada, S. Goy-López, A. Cambón, M.-B.
Madec, S. G. Yeates, V. Mosquera, J. Phys. Chem. B 2009,
113, 12391-12399.
43. C. N. Pace, F. Vajdos, L. Fee, G. Grimsley, T. Gray, Protein
Sci. 1995, 4, 2411-2423.
44. S. W. Provencher, Comp. Phys. Comm. 1982, 27, 213-227.
45. A. Lobley, L. Whitmore, B. A. Wallace, Bioinformatics 2002,
18, 211-212.
46. L. Whitmore, B. A. Wallace, Nucleic Acids Res. 2004, 32,
W668-W673.
47. M. J. Geisow, G. H. Beaven, Biochem. J. 1977, 163, 477-484.
48. B. Farruggia, F. Rodriguez, R. Rigatuso, G. Fidelio, G. Pico, J.
Protein Chem. 2001, 20, 81-89.
49. K. Flora, J. D. Brennan, G. A. Baker, M. A. Doody, F. V.
Bright, Biophys. J. 1998, 75, 1084-1096.
50. N. K. Holm, S. K. Jespersen, L. V. Thomassen, T. Y. Wolff, P.
Sehgal, L. A. Thomsen, G. Christiansen, C. B. Andersen, A.
D. Knudsen, D. E. Otzen, Biochim. Biophys. Acta 2007, 1774,
1128-1138.
51. F. Ferrone, W. Ronald, Analysis of protein aggregation
kinetics. In Methods in Enzymology, Academic Press: 1999,
309, 256-274.
52. M. Alatorre-Meda, P. Taboada, J. Sabin, B. Krajewska, L. M.
Varela, J. R. Rodriguez, Colloids Surfaces A 2009, 339, 145-
152.
53. P. Shahi, R. Sharma, S. Sanger, I. Kumar, R. S. Jolly,
Biochemistry 2007, 46, 7365-7373.
54. J. T. Jarrett, P. T. Lansbury, Biochemistry 1992, 31, 12345-
12352.
55. A. Lomakin, D. B. Teplow, D. A. Kirschner, G. B. Benedek,
Proc. Natl. Acad. Sci. USA 1997, 94, 7942-7947.
56. R. Bader, R. Bamford, J. Zurdo, B. F. Luisi, C. M. Dobson, J.
Mol. Biol. 2006, 356, 189-208.
57. Q. Xu, T. A. Keiderling, Biochemistry 2005, 44, 7976-7987.
58. A. J. Rader, B. M. Hespenheide, L. A. Kuhn, M. F. Thorpe,
Proc. Natl. Acad. Sci. USA 2002, 99, 3540-3545.
59. L. Nielsen, R. Khurana, A. Coats, S. Frokjaer, J. Brange, S.
Vyas, V. N. Uversky, A. L. Fink, Biochemistry 2001, 40,
6036-6046.
60. S. Goda, K. Takano, Y. Yamagata, R. Nagata, H. Akutsu, S.
Maki, K. Namba, K. Yutani, Protein Sci. 2000, 9, 369-375.
61. Y. Yonezawa, S. Tanaka, T. Kubota, K. Wakabayashi, K.
Yutani, S. Fujiwara, J. Mol. Biol. 2002, 323, 237-251.
62. N. A. P. Ulrih, G. Anderluh, P. Macek, T. V. Chalikian,
Biochemistry 2004, 43, 9536-9545.
63. M. Dockal, D. C. Carter, F. Ruker, J. Biol. Chem. 2000, 275,
3042-3050.
64. Y. Kumar, S. Tayyab, S. Muzammil, Archiv. Biochem.
140 Biophys. 2004, 426, 3-10.
196

5
65. L. A. Sikkink, M. Ramirez-Alvarado, Biophys. Chem. 2008,
135, 25-31.
66. I. I. Stepuro, E. A. Lapshina, N. A. Chaikovskaia, Mol Biol
(Mosk) 1991, 25, 337-347.
67. S. Grudzielanek, R. Jansen, R. Winter, J. Mol. Biol. 2005, 351,
879-894.
68. L. M. C. Sagis, C. Veerman, E. van der Linden, Langmuir
2004, 20, 924-927.
69. Y. Fezoui, D. B. Teplow, J. Biol. Chem. 2002, 277, 36948-
10 36954.
25
70. K. Sasahara, H. Yagi, H. Naiki, Y. Goto, Y. J. Mol. Biol. 2007,
372, 981-991.
71. V. N. Uversky, A. L. Fink, Biochim. Biophys. Acta 2004,
1698, 131-153.
72. M. Bhattacharya, N. Jain, S. Mukhopadhyay, J. Phys. Chem. B
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 13
15
2011, 115, 4195-4205.
73. N. Hirota-Nakaoka, K. Hasegawa, H. Naiki, Y. Goto, J.
Biochem. 2003, 134, 159-164.
74. S. Jordens, J. Adamcik, I. Amar-Yuli, R. Mezzenga,
20 Biomacromolecules 2011, 12, 187-193.
197

Obtention of Metallic Nanowires by Protein
Biotemplating and Their Catalytic Application
Josue Juarez, Adriana Cambon, Sonia Goy-Lopez, Antonio Topete,
Pablo Taboada,* and Víctor Mosquera
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada, Facultad de Física,
Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
ABSTRACT Gold nanowires were obtained by a seeded growth process using
protein lysozyme fibrils as biotemplates. The degree of metal coverage until full
biotemplate coverage onto the fibril was controlled by the gold salt concentration
and the number of sequential additions of metal growth solutions. The hybrid
fibrils might have a potential use as catalysts since they display enhanced catalytic
activity in the reduction of p-nitrophenol to p-aminophenol by NaBH 4.
SECTION Nanoparticles and Nanostructures
Metal nanoparticles have received considerable attention
in past decade because of their particular optical,
electronic, magnetic, and catalytic properties and their
important applications in many fields such as nanosensors, 1-4
catalysis, 5-8 biomedicine, 9-11 biological labeling, 12-14 and
surface-enhanced Raman scattering (SERS). 15-18 To optimize
and extend the application of metal NPs, methods must be
developed to control the assembly and organization of these
nanomaterials. Assemblies of NPs provide optical and electronic
properties that are distinct compared to individual
particles or disorganized macroscale agglomeration. In this
regard, Nature offers us the possibility to take advantage of the
well-defined structures and special properties of biomolecules
and their supramolecular structures to organize nanoparticles
into predefined, topologically intricate nanostructures or to
synthesize miscellaneous materials in order to control the
properties of nanoparticle assemblies for potential applications
in electronic, optical, and chemical devices. 19-23 In
particular, functionalizing one-dimensional (1D) supporting
biomaterials with metal NPs that combine the properties of
two functional nanomaterials, such as high conductivity surface
area or precise chemical functionality of the biotemplate
and the unique plasmonic or catalytic properties of metal NPs,
to achieve a wider range of applications will therefore play an
important role in the development of nanoscience and nanotechnology.
As a result, considerable efforts have been directed
toward the use of 1D biotemplates such as DNA, 24-27
viruses, 28-31 peptides, 32-34 proteins, 35-38 or fungus 39 to construct
corresponding multifunctional hybrid 1D nanostructures
such as nanowires, 40-43 nanotubes, 44-46 or nanodumbbells. 47
The spontaneous fibrillation mechanism which different
proteins undergo under suitable conditions results in the
formation of amyloid-like fibrils, 37,48,49 which should be valid
as alternative templating agents for the construction of 1D
hybrid materials. In particular, lysozyme (Lys) is a protein
which is known to fibrillate under different solution conditions
pubs.acs.org/JPCL
to form protein fibrils with diameters ranging from 10 to 50 nm
and lengths up to several micrometers (see Figure 1a). 50-52
The dimensions of the fibrils can be tuned by varying the
solution conditions and the incubation time. They are also
thermally stable at high temperatures and different pHs, and
they contain multiple potential ion-binding sites within their
amino acid sequences, which enables their use in metallization
reactions under relatively harsh conditions. Thus, we
exploit all of these features to generate 1D nanoparticle
assemblies and nanowires by in situ nanoparticle formation
and seeding growth on the fibril surface and to analyze their
potential applicability as biocatalysts.
Although biotemplating of 1D metallic nanoparticle assemblies
and nanowires by peptides and protein fibrils has
been previously reported 35,37,38,40,43 and their electric and
magnetic properties have been evaluated, 35,53 less attention
has been paid to the obtention of 1D protein-fibril-templated
metallic nanostructures with a controlled degree of metallization
and their potential application as a catalyst. Here, we
present a seeded-mediated method for the controlled coverage
by Au NPs on the surface of the fibrils formed by the
protein Lys in order to find the optimal conditions for the use
of the present biohybrid systems as a catalyst. We test their
catalytic capability using the model reduction reaction of
p-nitrophenol by NaBH 4.
Lys fibrils were prepared as previously described (see
Experimental Section). Au-nanoparticle-coated protein fibrils
were obtained by the addition of preformed citrate-stabilized
Au seeds to an aqueous solution containing self-assembled
fibrils at pH 2.0. Provided that the isoelectric point of Lys is
around 11, 54 it is expected that negatively charged gold seed
Received Date: July 26, 2010
Accepted Date: August 23, 2010
r XXXX American Chemical Society 2680 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
199

ions would interact electrostatically with electrically charged
amine groups located at the fibril surfaces after incubation.
Subsequently, different sequential additions of a solution
containing HAuCl 4 and ascorbic acid were performed in order
to achieve a full coverage of the hybrid composites. Different
initial [Au seeds]/[protein] molar ratios (MR) were tested in
order to find an initial suitable MR to ensure full coverage after a
few additions and to avoid both precipitation of the nanohybrid
and the generation of free gold NPs. An initial [Au seed]/[protein]
MR=50wasfoundtobetheoptimaloneandwasfurtherused
during the sequential additions of Au ions to achieve the
formation of the metallic nanowires (see Table 1).
The morphologies of the resulting products were investigated
by transmission electron microscopy (TEM) and scanning
pubs.acs.org/JPCL
Figure 1. TEM images of (a) bare Lys fibrils and Au-fibril biohybrids obtained after (b) zero, (c) one, (d) two, (e) three, and (f) four sequential
additions of the Au growth solution; (g) bright field and (h) dark field TEM images of fully covered fibrils; (i) SEM image of a Au-metallic
biohybrid nanowire. Scale bars are (a,i) 500, (b) 100, and (c-h) 200 nm.
Table 1. Au Content of the Obtained Nanohybrids
sample ([Au]/[protein] MR) Au concentration (10 -5 M)
biohybrid 1(50) 3.4
biohybrid 2 (100) 3.9
biohybrid 3 (125) 4.6
biohybrid 4 (150) 5.3
biohybrid 5 (200) 6.0
transmission microscopy (SEM). Figure 1a shows a typical
TEM image of the as-prepared Lys fibrils, with lengths
ranging from 0.4 to 8 μm and widths of 12-20 nm. The
metallic coverage of the nanofibrils could be easily controlled
by changing the number of sequential additions of
gold growth solution into the fibril solution over already
preformed Au nanocrystals. Figure 1b-e shows the progressive
coverage of the nanofibrils after increasing the number
of sequential additions, with the formation of more metallic
nanoparticles onto the fibril surface. TEM revealed that
metallic nanoparticles on the fibril surfaces possess a size
ranging from 3.7 to 8.0 ( 2.0 nm, depending on the number
of sequential additions of the gold growth solution. The
continuous coverage is a result of the growth of pre-existing
NPs and generation of new ones on the fibril surface and
subsequent fusion of adjacent as-synthesized NPs. The fully
covered fibrils display a relatively rougher surface than
pristine and partially covered fibrils, as can be observed
from the TEM image obtained in the backscattering mode
and the SEM picture obtained without the need for metallic
contrast (see Figure 1h,i). Also, the size of the metallic NPs
clearly increases, and the size population can also broadened
(see Figure S1, Supporting Information). In addition,
r XXXX American Chemical Society 2681 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
200

Figure 2. (a) XRD pattern of spherical Au NPs on the surface of protein fibrils; (b) EDX pattern of a gold hybrid fibril.
under conditions of excessive addition of Au ions in solution,
an increasing presence of fibril networks is observed as a
consequence of fibril bundling, which leads to precipitation
of the nanowires after several days (see Figure S2, Supporting
Information).
The formation of hybrid fibrils was further characterized by
energy dispersive X-ray (EDX) spectroscopy and X-ray diffraction
(XRD). XRD patterns of the resulting hybrid fibrils display
the classical peaks corresponding to metallic spherical gold
NPs. The crystallite sizes calculated according to the modified
Scherrer relation 55 for the hybrid fibrils range from 5 to 8 nm
depending on the number of additions of Au growth solution.
The EDX spectrum (Figure 2b) of protein-fibril-supported Au
NPs shows the peaks corresponding to C, O, S, and Au,
confirming the existence of Au NPs on the surface of the Lys
fibrils. The S peak is characteristic of protein samples. 56
To study the performance of the Au NP hybrid fibrils as
catalysts, we have chosen the borohydride reduction of
p-nitrophenol as a model reaction. It is well documented that
this reaction can be catalyzed by noble metal nanoparticles,
and the color changes involved in the reduction also provide a
simple way based on spectroscopic measurements for monitoring
the reaction kinetics. 57,58 Under a neutral or acidic
condition, p-nitrophenol solution exhibits a strong absorption
peak at 317 nm. Upon the addition of NaBH4, the alkalinity of
pubs.acs.org/JPCL
the solution increases, and p-nitrophenolate ions would
become the dominating species, together with a spectral shift
to 400 nm for the absorption peak. 59,60 This peak remains
unaltered with time, suggesting that the reduction did not take
place in the absence of a catalyst, as reported elsewhere. 61,62
However, the addition of a small amount of the Au-based
catalyst (i.e., metal hybrid fibrils with a Au concentration
ranging from 0.002 to 0.02 mM) to the above reaction
mixture causes fading and ultimate bleaching of the yellow
color of the reaction mixture quickly. Time-dependent absorption
spectra of this reaction mixture show the disappearance
of the peak at 400 nm that is accompanied by a gradual
development of a new peak at 300 nm corresponding to the
formation of p-aminophenol (Figure 3a). The UV spectra also
exhibit an isosbestic point between two absorption bands,
indicating that only two principal species, p-nitrophenol and
p-aminophenol, influence the reaction kinetics. Similar spectral
changes were also observed with other hybrid fibrils with
different Au NPconcentrations. These results indicate that the
hybrid fibrils can successfully catalyze the reduction reaction.
Several research groups have also reported similar kinds of
spectral changes of p-nitrophenol during its catalytic reduction
in the presence of both supported and unsupported
spherical metallic nanoparticles. 63-65 In this reduction process,
as the concentration of NaBH4 in the reaction mixture far
r XXXX American Chemical Society 2682 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
201

pubs.acs.org/JPCL
Figure 3. (a) Time evolution of UV-vis spectra and (b) variation of absorbance with time upon conversion of p-nitrophenol to
p-aminophenol in the presence of biohybrid 2 with Au concentrations of (0) 0.26, (b) 0.53, (4) 0.79, (1) 1.05, and (þ) 1.31 10 -5 M.
The arrow in (a) denotes the decreasing presence of p-nitrophenol in the mixture as the reduction reaction proceeds. Variation of (c) k app
and (d) k nor with Au concentration present in the reduction reaction: (9) biohybrid 1, (O) biohybrid 2, (2) biohybrid 3,; (þ) biohybrid 4, and
(3) biohybrid 5.
exceeds the concentration of p-nitrophenol, the reaction rate
is assumed to follow first-order kinetics. Keeping this in mind,
we decided to measure the concentrations of p-nitrophenolate
ions and thus to monitor the progress or kinetics of the
reaction by recording the absorbance at 400 nm because the
latter peak was much stronger than that at 315 nm. We
calculated the values of the apparent rate constants (kapp) of
the catalytic reaction in the presence of different concentrations
of the hybrid fibrils possessing different degrees of Au
coverage from the change of absorbance with time (Figures 3b
and S3, Supporting Information). These plots are straight
lines, indicating that the reduction reaction effectively follows
first-order kinetics. The k app values of the hybrid fibrils
rise linearly with an increase of their concentration (and,
thus, also with increases in Au concentration) in the reaction
medium until reaching a maximum to decrease further (see
Figure 3c). In addition, values of k app for p-nitrophenol
reduction using hybrid fibrils were ∼1.5-4 times larger
than those reported for glucose-reduced Au nanoparticle
networks, 64 poly(amidoamine) (PAMAM)-dendrimer-supported
spherical nanoparticles, 59 solid 1D Au nanobelts 66
and nanorods, 67 polymer-micelle-supported Au NPs, 68,69 multicomponent
microgels, 70 and 1D assemblies of Au NPs, 71 and
they were of similar order as those obtained for porous 1D
nanobelts 66 or Au yolk-shell NPs. 72 Nevertheless, we have to
note that we used very small amounts of gold catalysts in our
experiments.
Thus, to get a clearer picture of the efficacy of the composite
material as a catalyst and also to compare the catalytic
activity between hybrid fibrils with different degrees of coverage,
the catalytic activities are also presented in terms of a
normalized rate constant (knor) obtained by normalization
of k app to the total amount (mmol) of catalyst (gold) used in
the reaction. Figure 3d shows that the values of k nor for
p-nitrophenol reduction in the presence of the different types
of hybrid fibrils prepared at different [Au]/[protein] MR values
are also different. In particular, k nor remains relatively constant
from hybrid 1 to 3, that is, as the coverage of the fibrils
increases, while for hybrids 4 and 5, k nor progressively
reduces. This behavior might be a consequence of (i)
changes in nanoparticle sizes on the fibril surfaces and (ii)
a decrease in the available metal surface area for the
catalytic reaction to take place as the fibril coverage is almost
complete. In this regard, it is well-known that the catalytic
activity increases as the size of Au NPs decreases because of
the increase of effective surface area. In our case, although
the crystallite size increases as the concentration of the
growth solution increases, we have noted a larger increase
in nanoparticle mean size accompanied with an important
polydispersity for nanohybrids 4 and 5, as previously
r XXXX American Chemical Society 2683 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
202

(15) Banholzer, M. J.; Millstone, J. E.; Qin, L.; Mirkin, C. A. Rationally
Designed Nanostructures for Surface-Enhanced Raman
Spectroscopy. Chem. Soc. Rev. 2008, 37, 885–897.
(16) Li, W.; Camargo, P. H. C.; Lu, X.; Xia, Y. Dimers of Silver
Nanospheres: Facile Synthesis and Their Use as Hot Spots for
Surface-Enhanced Raman Scattering. Nano Lett. 2009, 9,
485–490.
(17) Rodríguez-Lorenzo, L.; Alvarez-Puebla, R. A.; Pastoriza-Santos,
I.; Mazzucco, S.; Stephan, O.; Kociak, M.; Liz-Marzan, L. M.;
García de Abajo, F. J. Zeptomol Detection Through Controlled
Ultrasensitive Surface-Enhanced Raman Scattering. J. Am.
Chem. Soc. 2009, 131, 4616–4618.
(18) Alvarez-Puebla, R. A.; Liz-Marzan, L. M. SERS-Based Diagnosis
and Biodetection. Small 2010, 6, 604–610.
(19) van Bommel, K. J. C.; Friggeri, A.; Shinkai, S. Organic
Templates for the Generation of Inorganic Materials. Angew.
Chem., Int. Ed. 2003, 42, 980–999.
(20) Baron, R.; Willner, B.; Willner, I. Biomolecule-Nanoparticle
Hybrids as Functional Units for Nanobiotechnology. Chem.
Commun. 2007, 323–332.
(21) Ofir, Y.; Samanta, B.; Rotello, V. M. Polymer and Biopolymer
Mediated Self-Assembly of Gold Nanoparticles. Chem. Soc.
Rev. 2008, 37, 1814–1825.
(22) Sotiropoulou, S.; Sierra-Sastre, Y.; Mark, S. S.; Batt, C. A.
Biotemplated Nanostructured Materials. Chem. Mater. 2008,
20, 821–834.
(23) Behrens, S. S. Synthesis of Inorganic Nanomaterials
Mediated by Protein Assemblies. J. Mater. Chem. 2008, 18,
3788–3798.
(24) Warner, M. G.; Hutchison, J. E. Linear Assemblies of Nanoparticles
Electrostatically Organized on DNA Scaffolds. Nat.
Mater. 2003, 2, 272–277.
(25) Gun, Q.; Chen, C.; Haynie, D. T. Cobalt Metallization of DNA:
Toward Magnetic Nanowires. Nanotechnology 2005, 16,
1358–1363.
(26) Kundu, S.; Liang, H. Photochemical Synthesis of Electrically
Conductive CdS Nanowires on DNA Scaffolds. Adv. Mater.
2008, 20, 826–831.
(27) Wirges, C. T.; Timper, J.; Fischler, M.; Sologubenko, A. S.;
Mayer, J.; Simon, U.; Carell, T. Controlled Nucleation of DNA
Metallization. Angew. Chem., Int. Ed. 2009, 48, 219–223.
(28) Knez, M.; Bittner, A. M.; Boes, F.; Wege, C.; Jeske, H.; Maiβ, E.;
Kern, K. Biotemplate Synthesis of 3-nm Nickel and Cobalt
Nanowires. Nano Lett. 2003, 3, 1079–1082.
(29) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J.; Mann, S. Organization
of Metallic Nanoparticles Using Tobacco Mosaic Virus
Templates. Nano Lett. 2003, 3, 413–417.
(30) Tsukamoto, R.; Muraoka, M.; Seki, M.; Tabata, H.; Yamashita,
I. Synthesis of CoPt and FePt3 Nanowires Using the Central
Channel of Tobacco Mosaic Virus as a Biotemplate. Chem.
Mater. 2007, 19, 2389–2391.
(31) Balci, S.; Leinberger, D. M.; Knez, M.; Bittner, A. M.; Boes, F.;
Kadri, A.; Wege, C.; Jeske, H.; Kern, K. Printing and Aligning
Mesoscale Patterns of Tobacco mosaic Virus on Surfaces. Adv.
Mater. 2008, 20, 2195–2200.
(32) Fu, X.; Wang, Y.; Huang, L.; Sha, Y.; Gui, L.; Lai, L.; Tang, Y.
Assemblies of Metal Nanoparticles and Self-Assembled Peptide
Fibrils ; Formation of Double Helical and Single-Chain
Arrays of Metal Nanoparticles. Adv. Mater. 2003, 15,902–906.
(33) Gazit, E. Self-Assembled Peptide Nanostructures: the Design
of Molecular Building Blocks and Their Technological Utilization.
Chem. Soc. Rev. 2007, 36, 1263–1269.
(34) Sharma, N.; Top, A.; Kiick, K. L.; Pochan, D. J. One-Dimensional
Gold Nanoparticle Arrays by Electrostatically Directed
pubs.acs.org/JPCL
Organization Using Polypeptide Self-Assembly. Angew.
Chem., Int. Ed. 2009, 48, 7078–7082.
(35) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger,
H.; Lindquist, S. L. Conducting Nanowires Built by Controlled
Self-Assembly of Amyloid Fibers and Selective Metal Deposition.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527–4532.
(36) Behrens, S.; Wu, J.; Habitch, W.; Unger, E. Silver Nanoparticle
and Nanowire Formation by Microtubule Templates. Chem.
Mater. 2004, 16, 3085–3090.
(37) Cherny, I.; Gazit, E. Amyloids: Not Only Pathological Agents
but Also Ordered Nanomaterials. Angew. Chem., Int. Ed. 2008,
47, 4062–4069.
(38) Slocik, J. M.; Kim, S. N.; Whitehead, T. A.; Clark, D. S.; Naik,
R. R. Biotemplated Metal Nanowires Using Hyperthermophilic
Protein Filaments. Small 2009, 5, 2038–2042.
(39) Bigall, N. C.; Reitzing, M.; Naumann, W.; Simon, P.; van Pee,
K.-H.; Eychm€uller, A. Fungal Templates for Noble-Metal
Nanoparticles and Their Application in Catalysis. Angew.
Chem., Int. Ed. 2008, 47, 7876–7879.
(40) Retches, M.; Gazit, E. Casting Metal Nanowires Within Discrete
Self-Assembled Peptide Nanotubes. Science 2003, 300,
625–627.
(41) Kinsella, J. M.; Ivanisevic, A. Enzymatic Clipping of DNA
Wires Coated with Magnetic Nanoparticles. J. Am. Chem.
Soc. 2005, 127, 3276–3277.
(42) Bai, H.; Xu, K.; Xu, Y.; Matsui, H. Fabrication of Au Nanowires
of Uniform Length and Diameter Using a Monodisperse and
Rigid Biomolecular Template: Collagen-like Triple Helix. Angew.
Chem., Int. Ed. 2007, 46, 3319–3322.
(43) Ostrov, N.; Gazit, E. Genetic Engineering of Biomolecular
Scaffolds for the Fabrication of Organic and Metallic Nanowires.
Angew. Chem., Int. Ed. 2010, 49, 3018–3021.
(44) Guha, S.; Banerjee, A. Self-Assembled Robust Dipeptide
Nanotubes and Fabrication of Dipeptide-Capped Gold Nanoparticles
on the Surface of these Nanotubes. Adv. Funct. Mater.
2009, 19, 1949–1961.
(45) Ryu, J.; Lim, S. Y.; Park, C. B. Photoluminescent Peptide
Nanotubes. Adv. Mater. 2009, 11, 1577–1581.
(46) Gorzny, M. L.; Walton, A. S.; Evans, S. D. Synthesis of High-
Surface-Area Platinum Nanotubes Using a Viral Template.
Adv. Funct. Mater. 2010, 20, 1295–1300.
(47) Balci, S.; Noda, K.; Bittner, A.; Kadri, A.; Wege, C.; Jeske, H.;
Kern, K. Self-Assembly of Metal-Virus Nanodumbbells. Angew.
Chem., Int. Ed. 2007, 46, 3149–3151.
(48) Dobson, C. M. Protein Folding and Misfolding. Nature 2003,
426, 884–890.
(49) Jahn, T. R.; Radford, S. E. Folding Versus Aggregation: Polypeptide
Conformations on Competing Pathways. Arch. Biochem.
Biophys. 2008, 469, 100–117.
(50) Frare, E.; De Laureto, P. P.; Zurdo, J.; Dobson, C. M.; Fontana,
A. A Highly Amyloidogenic Region of Hen Lysozyme. J. Mol.
Biol. 2004, 340, 1153–1165.
(51) Pawar, A. P.; Dubai, K. F.; Zurdo, J.; Chiti, F.; Vendruscolo,
M.; Dobson, C. M. Prediction of “Aggregation-Prone” and
“Aggregation-Susceptible” Regions in Proteins Associated
with Neurodegenerative Diseases. J. Mol. Biol. 2005, 350,
379–392.
(52) Lieu, V. H.; Wu, J. W.; Wang, S. S. S.; Wu, C. H. Inhibition
of Amyloid Fibrillization of Hen Egg-White Lysozymes by
Rifampicin and p-Benzoquinone. Biotechnol. Prog. 2007, 23,
698–706.
(53) Yu, L.; Banerjee, I. A.; Shima, A.; Rajan, K.; Matsui, H. Size-
Controlled Ni Nanocrystal Growth on Peptide Nanotubes and
Their Magnetic Properties. Adv. Mater. 2004, 16, 709–712.
r XXXX American Chemical Society 2686 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
205

(54) Almeida, N. L.; Oliveira, C. L. P.; Torriani, I. L.; Loh, W.
Calorimetric and Structural Investigation of the Interaction
of Lysozyme and Bovine Serum Albumin with Poly(Ethylene
Oxide) and its Copolymers. Colloids Surf., B 2004, 38,67–76.
(55) Dutta, P.; Manivannan, A.; Seehra, M. S.; Shah, N.; Huffman,
G. P. Magnetic Properties of Nearly Defect-Free Maghemite
Nanocrystals. Phys. Rev. B 2004, 70, 174428/1–174428/7.
(56) Shiomi, T.; Tsunoda, T.; Kawai, A.; Mizukami, F.; Sakaguchi, K.
Biomimetic Synthesis of Lysozyme-Silica Hybrid Hollow
Particles Using Sonochemical Treatment: Influence of pH
and Lysozyme Concentration on Morphology. Chem. Mater.
2007, 19, 4486–4493.
(57) Schrinner, M.; Ballauff, M.; Talmon, Y.; Kauffmann, Y.; Thun,
J.; Moller, M.; Breu, J. Dynamical Quorum Sensing and
Synchronization in Large Populations of Chemical Oscillators.
Science 2009, 323, 617–620.
(58) Qin, G.; W. Pei, W.; Ma, X.; Xu, X.; Ren, Y.; Sun, W.; Zuo, L.
Enhanced Catalytic Activity of Pt Nanomaterials: From
Monodisperse Nanoparticles to Self-Organized Nanoparticle-Linked
Nanowires. J. Phys. Chem. C 2010, 114, 6909–
6913.
(59) Hayakawa, K.; Yoshimura, T.; Esumi, K. Preparation of Gold-
Dendrimer Nanocomposites by Laser Irradiation and Their
Catalytic Reduction of 4-Nitrophenol, Immobilization and
Recovery of Au Nanoparticles from Anion Exchange Resin:
Resin-Bound Nanoparticle Matrix as a Catalyst for the Reduction
of 4-Nitrophenol. Langmuir 2003, 19, 5517–5521.
(60) Praharaj, S.; Nath, S.; Gosh, S. K.; Kundu, S.; Pal, T. Immobilization
and Recovery of Au Nanoparticles from Anion Exchange
Resin: Resin-Bound Nanoparticle Matrix as a Catalyst
for the Reduction of 4-Nitrophenol. Langmuir 2004, 20,
9889–9892.
(61) Rashid, M. H.; Bhattacharjee, R. R.; Kotal, A.; Mandal, T. K.
Synthesis of Spongy Gold Nanocrystals with Pronounced
Catalytic Activities. Langmuir 2006, 22, 7141–7143.
(62) Rashid, M. H.; Mandal, T. K. Organic Ligand-Mediated Synthesis
of Shape-Tunable Gold Nanoparticles: An Application of
Their Thin Film as Refractive Index Sensors. J. Phys. Chem. C
2007, 111, 9684–9693.
(63) Esumi, K.; Isono, R.; Yoshimura, T. Preparation of PAMAMand
PPI-Metal (Silver, Platinum, and Palladium) Nanocomposites
and Their Catalytic Activities for Reduction of
4-Nitrophenol. Langmuir 2004, 20, 237–243.
(64) Liu, J.; Qin, G.; Raavendran, P.; Ikushima, Y. Facile “Green”
Synthesis, Characterization, and Catalytic Function of β-D-
Glucose-Stabilized Au Nanocrystals. Chem.;Eur. J. 2006, 12,
2131–2138.
(65) Chen, X.; Zhao, D.; An, Y.; Shi, L.; Hou, W.; Chen, L. Catalytic
Properties of Gold Nanoparticles Immobilized on the Surfaces
of Nanocarriers. J. Nanopart. Res. 2010, 12, 1877–1887.
(66) Li, L.; Wang, Z.; Huang, T.; Xie, J.; Qin, L. Porous Gold
Nanobelts Templated by Metal-Surfactant Complex Nanobelts.
Langmuir 2010, 26, 12330–12335.
(67) Bai, X.; Gao, Y.; Liu, H.-g.; Zheng, L. Synthesis of Amphiphilic
Ionic Liquids Terminated Gold Nanorods and Their Superior
Catalytic Activity for the Reduction of Nitro Compounds.
J. Phys. Chem. C 2009, 113, 17730–17736.
(68) Wang, Y.; Wei, G.; Zhang, W.; Jiang, X.; Zheng, P.; Shi, L.;
Dong, A. Responsive Catalysis of Thermoresponsive Micelle-
Supported Gold Nanoparticles. J. Mol. Catal. A: Chem. 2007,
266, 233–238.
(69) Chen, X.; An, Y.; Zhao, D.; He, Z.; Zhang, Y.; Cheng, J.; Shi, L.
Core-Shell-Corona Au-Micelle Composites with a Tunable
Smart Hybrid Shell. Langmuir 2008, 24, 8198–8204.
pubs.acs.org/JPCL
(70) Hain, J.; Schrinner, M.; Lu, Y.; Pich, A. Design of Multicomponent
Microgels by Selective Deposition of Nanomaterials.
Small 2008, 4, 2016–2024.
(71) Huang, T.; Meng, F.; Qi, L. Facile Synthesis and One-Dimensional
Assembly of Cyclodextrin-Capped Gold Nanoparticles
and Their Applications in Catalysis and Surface-Enhanced
Raman Scattering. J. Phys. Chem. C 2009, 113, 13636–13642.
(72) Lee, J.; Park, L. C.; Song, H. A Nanoreactor Framework of
a Au@SiO 2 Yolk/Shell Structure for Catalytic Reduction of
p-Nitrophenol. Adv. Mater. 2008, 20, 1523–1528.
(73) Rashid, M. H.; Mandal, T. K. Templateless Synthesis of
Polygonal Gold Nanoparticles: An Unsupported and Reusable
Catalyst with Superior Activity. Adv. Funct. Mater. 2008, 18,
2261–2271.
(74) Kumar, S. S.; Kumar, C. S.; Mathiyarasu, J.; Phani, K. L.
Stabilized Gold Nanoparticles by Reduction Using 3,4-Ethylenedioxythiophene-polystyrenesulfonate
in Aqueous Solutions:
Nanocomposite Formation, Stability, and Application
in Catalysis. Langmuir 2007, 23, 3401–3408.
(75) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chemistry
and Properties of Nanocrystals of Different Shapes. Chem.
Rev. 2005, 105, 1025–1102.
(76) Knubovets, T.; Osterhout, J. J.; Connolly, P. J.; Klibanov, A. M.
Structure, Thermostability, And Conformational Flexibility of
Hen Egg-White Lysozyme Dissolved in Glycerol. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 1262–1267.
r XXXX American Chemical Society 2687 DOI: 10.1021/jz101029f |J. Phys. Chem. Lett. 2010, 1, 2680–2687
206

DOI: 10.1002/chem.201003679
One-Dimensional Magnetic Nanowires Obtained by Protein Fibril
Biotemplating
JosuØ Juµrez, Adriana Cambón, Antonio Topete, Pablo Taboada,* and
Víctor Mosquera [a]
Abstract: Magnetic nanowires were obtained
through the in situ synthesis of
magnetic material by Fe-controlled
nanoprecipitation in the presence of
two different protein (human serum albumin
(HSA) and lysozyme (Lys)) fibrils
as biotemplating agents. The
structural characteristics of the biotemplates
were transferred to the hybrid
magnetic wires. They exhibited excel-
Introduction
A key issue in nanotechnology is the development of conceptually
simple construction techniques for the mass fabrication
of identical nanoscale structures. Interest in one-dimensional
(1D) nanoscale materials and devices, often
called nanowires, nanotubes or nanorods, has risen sharply
in recent years. In particular, 1D magnetic nanostructures
exhibit unique magnetic properties due to geometric confinement,
magnetostatic interactions and nanoscale domain
formation, [1] which endow them with potential applicability
in data storage and logic devices, [2a] magneto-transport behaviour,
[2b] micromechanical sensors [2c] and biomedicine. [2d]
As a result of their high aspect ratios, 1D magnetic entities
possess larger dipole moments than individual nanoparticles.
This allows their manipulation with lower field strengths [3]
and provides them with improved imaging contrast capabilities,
which opens up the possibility of their use in new biomedical
applications, specifically in the advent of low-field
MRI. [4] Here we report the formation of 1D magnetic nanowires
and assemblies of magnetic nanoparticles over linear
nanosized biopolymer templates formed by spontaneous fibrillation,
under suitable conditions, of two proteins—
human serum albumin (HSA) and lysozyme (Lys)—by in
situ co-precipitation of iron under suitable conditions. These
[a] J. Juµrez, A. Cambón, A. Topete, P. Taboada, V. Mosquera
Grupo de Física de Coloides y Polímeros
Departamento de Física de la Materia Condensada
Facultad de Física, Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
Fax: (+ 34) 881814112
E-mail: pablo.taboada@usc.es
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003679.
7366
lent magnetic properties as a consequence
of the 1D assembly and fusion
of magnetite nanoparticles as ascertained
by SQUID magnetometry.
Prompted by these findings, we also
Keywords: biotemplating · imaging
agents · magnetic nanowires · nanostructures
· protein fibrils
checked their potential applicability as
MRI contrast agents. The magnetic
wires exhibited large r 2* relaxivities
and sufficient contrast resolution even
in the presence of an extremely small
amount of Fe in the magnetic hybrids,
which would potentially enable their
use as T 2 contrast imaging agents.
magnetic 1D nanostructures possess both high saturation
magnetisations and spin–spin relaxivities at low magnetic
fields, which makes them suitable candidates for use as
imaging contrast agents in MRI.
There are many literature reports on the fabrication of
1D magnetic nanostructures, which can be assembled by (bio)templating,
spontaneous self-assembly by magnetic dipolar
interactions of nanoparticles, chemical synthesis, lithographic
methods, laser etching or microcontact printing. [5]
Owing to its relative simplicity, template-directed synthesis
is one of the most attractive methods for preparing magnetic
1D nanomaterials. [5a,b] This approach offers exciting alternatives
to the costly nanolithography-based “top-down” technologies
or multi-step chemical procedures. Various linear
nanometer-scale materials, including molecular dispersed
and assembled polymers, [6] carbon nanotubes [5a,7] or biological
scaffolds, have been employed to prepare 1D magnetic
structures and assemblies. [8] In particular, the capability of
biopolymers to generate 1D magnetic nanostructures is
really exciting because nature provides a renewable and
highly diverse source of nanometer-scale ordered complexes
that can be used to replicate inorganic materials as well-defined
structures. [9] Dextran, for example, has been used in
the creation of elongated assemblies of spherical nanoparticles
suitable for use as MRI contrast agents and tumour targeting
species. [2d, 10] DNA was also used as a stabiliser for the
formation of ordered nanowires of magnetic nanoparticles.
[2b, 11] These nanowire assemblies resulted in stable magnetic
fluids with high relaxivities at low fields useful for
MRI imaging. The protein cage of cowpea chlorotic mottle
virus (CCMV) was explored with the same goal, to incorporate
high payloads of Gd 3+ with elevated molecular relaxivities,
[12] and the formation either of magnetic nanotubes or of
magnetic nanowires with the aid of magnetic bacteria (Mag-
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 7366 – 7373

netospirillum magneticum strain AMB-1), [13] and tobacco
mosaic virus [14] were also investigated.
The spontaneous fibrillation undergone by different proteins
under suitable conditions results in the formation of
amyloid-like fibrils, [15] potentially applicable as alternative
templating agents for the construction of 1D magnetic materials.
In particular, the proteins HSA and Lys are two proteins
known to fibrillate under different solution conditions
to form fibrils with diameters ranging from 10 to 50 nm and
lengths up to several microns [16] (see Figure S1 in the Supporting
Information). The dimensions of the fibrils can be
tuned by varying the solution conditions and the incubation
times. They are also thermally stable at high temperatures
and different pH values and they contain multiple potential
ion-binding sites within their amino acid sequences, which
enables their use in metallisation reactions under relatively
harsh conditions. We have thus exploited these features to
generate 1D magnetic nanoparticle assemblies and nanowires
by in situ nanoparticle formation on the fibril surface
by use of the electrostatic interaction between Fe 2 + and
Fe 3 + ions and the negatively charged amino acid residues located
on the protein surfaces (mainly glutamic and aspartic
acids) [17] and analysis of their magnetic properties and potential
applicability as MRI contrast agents. Although biotemplating
of metallic nanowires by peptides and protein fibrils
has been reported previously, [8a, 18] less attention has
been paid to the production of 1D protein fibril-templated
magnetic structures, and their magnetic properties have not
been analysed in detail. Bolaamphiphile peptide fibrils, for
example, have been used to template the growth and size of
nickel nanocrystals on their surfaces and to tune their magnetic
properties, [19] self-assembled peptides have also been
conjugated to gadolinium ions, [20] and iron bionanomineralisation
has been found to occur in human serum transferrin
fibrils. [21]
Results and Discussion
Nanowire synthesis and characterisation: For in situ magnetic
nanoparticle formation and subsequent fibril coverage,
nanoparticle-coated protein fibrils were obtained by sequential
addition of Fe 2 + and Fe 3 + ions (1:2 molar ratio) and ammonium
hydroxide (NH 4OH) solutions to aqueous solutions
containing self-assembled fibrils, with subsequent incubation
at 80 8C (see the Experimental Section for protein fibrillation
and nanoparticle growth details). The controlled precipitation
of iron ions leads to the formation of magnetic nanoparticles
on the fibrils (see Figure 1). The effects of iron
concentration, iron/protein molar ratio, number of sequential
additions and/or the presence of different reagents/stabilisers
were tested to provide progressive full coverage of
the biotemplate and to generate a magnetic wire. A protocol
composed of sequential additions of Fe ions in several steps
(seeded-growth mechanism) was therefore performed (see
the Experimental Section). A suitable [Fe]/ACHTUNGTRENUNG[protein] molar
ratio (MR) was necessary to ensure full coverage after a
FULL PAPER
Figure 1. TEM images showing the progressive coverage of lysozyme fibrils
to afford magnetic nanowires after a single addition of Fe ions at
[Fe]/ACHTUNGTRENUNG[protein] molar ratios of: a) 25, b) 50, c) 100 and d) 100 together
with one additional sequential addition of Fe ions at the same molar
ratio. e) Size distributions of as-synthesised magnetic nanoparticles on
the biotemplate surfaces. The dotted line represents the fit of the distribution.
small number of addition steps and to avoid both precipitation
of the nanohybrid and generation of free magnetic NPs.
We should note that if the [Fe]/ACHTUNGTRENUNG[protein] molar ratio (MR =
10, 25, 50) is low, complete fibril coverage is not achieved
even after several sequential additions; in contrast, if the
[Fe]/ACHTUNGTRENUNG[protein] MR is too large, bundling and precipitation of
the nanohybrids occurs (see Figure S2 in the Supporting Information).
For these reasons an initial [Fe]/ACHTUNGTRENUNG[protein] MR=
100 was found to be optimal and was further used for three
sequential additions of Fe ions to achieve the formation of
the magnetic wires. Figure 1 a–d shows the progressive coverage
of the nanofibrils at different [Fe]/ACHTUNGTRENUNG[protein] molar
ratios, with the formation of magnetic nanoparticles on the
fibril surface. TEM revealed the primary magnetic nanoparticles
to be fairly uniformly distributed on the biotemplate
and to have sizes of about 6.8 2.0 nm with a relatively
narrow size distribution (Figure 1 e), in fair agreement with
those magnetic particles obtained in the absence of the protein
template (see Figure S3 in the Supporting Information).
Chem. Eur. J. 2011, 17, 7366 – 7373 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 7367

Also, it is necessary to note that fibril integrity and stability
was perfectly maintained under these reaction conditions
(see Figure S4 in the Supporting Information).
With more addition steps carried out the fibrils became
progressively covered with more magnetic material without
any apparent increase in nanoparticle size. The continuous
fibril coverage is a result of the fusion of adjacent as-synthesised
magnetic nanoparticles (see Figure S5 in the Supporting
Information). When a progressive and sufficient
excess of Fe was added, the nanowire widths increased and
their surfaces also appeared rougher, which might indicate
that more than one layer of magnetic material had been deposited
onto the template (Figure 2 a–b). In this regard, the
Figure 2. TEM images showing the progressive increases in the widths of
the magnetic wires and subsequent bundling after a) three, b) four or
c) five sequential additions of Fe ions at MR = 100. d) After 5 sequential
additions at MR = 100 in the presence of sodium citrate as stabiliser.
widths of the magnetic wires could be modified from 6 to
50 nm by increasing the number of sequential additions of
Fe ions onto the biotemplate. These values depend on the
type of fibril used as the template, the [Fe]/ACHTUNGTRENUNG[protein] molar
ratio and the number of sequential additions performed (see
Figure 1 and Figure S2 in the Supporting Information). Nevertheless,
constant width along the whole nanofibrils could
not be achieved, due to the non-uniform deposition of the
ions onto the template. We are currently modifying the synthetic
protocol with the goals both of improving uniformity
in fibril coverage and of achieving perfect control over the
magnetic nanowire dimensions and their resulting magnetic
properties. In addition, under conditions of excessive Fe
ions in solution an increasing presence of fibril networks as
a consequence of fibril bundling was observed, leading to
precipitation of the nanowires after several days (see Figure
2 c). This can be a result either of magnetic interaction
between nanowires or of inefficient stabilisation of the 1D
7368
P. Taboada et al.
nanostructures. To prevent these phenomena, we decided to
stabilise the magnetic nanowires further by the addition of
sodium citrate, which has been shown to be an effective stabiliser
for water-soluble magnetic nanoparticles. [22] In this
process, no fibril precipitation was observed for at least one
month and a lower level of bundling was confirmed, probably
as a consequence of the generation of a surface organic
layer that moderately screens the magnetic interactions between
magnetic covered fibrils (Figure 2 d).
On the other hand, the morphologies and sizes of the
fully covered fibrils made of HSA and Lys present some differences
irrespective of the method used to create the magnetic
nanowires. This is a consequence of the inherent structural
differences between the fibrils assembled from the two
proteins. In this respect, HSA-derived nanowires have shorter
lengths (between 0.1 and 2 mm) and widths (4–10 nm),
and have a curly morphology as a direct consequence of the
template structure (see Figure S6 in the Supporting Information).
[16a,b] In contrast, Lys-derived wires appear to be
straighter and longer (lengths and widths of 0.4–8 mm and
12–20 nm, respectively). [16c,d] This observation additionally
confirms that the overall structures of the fibril templates
remain intact after surface modification.
XRD analysis, selected area electron diffraction (SAED)
patterns, high-resolution TEM (HR-TEM) images and
FTIR spectroscopic examination of the fully covered fibrils
confirmed the formation and nature of the magnetic coverage
on the template surface. The XRD pattern showed reflections
indexed to (111), (220), (311), (222), (400), (422),
(511), (440) and (533) planes, corresponding to the cubic
spinel crystal structure of iron oxides (see Figure 3 a, JPCDS
file No. 19-0629, magnetite, or JCPDS file 39-1346, maghemite).
In addition, XRD demonstrated a polycrystalline
structure with cell constant a 0 = 0.8381 nm, which is in good
agreement with the standard card of iron oxides. The peak
broadening of the XRD pattern also indicates that the resulting
iron oxide crystallites were rather small. The crystallite
sizes calculated from the modified Scherrer relation [23]
for the magnetic fibrils of both Lys and HSA were about
(6.2 2) nm, consistent with the size distributions measured
from TEM images. SAED, HR-TEM and FTIR confirm the
predominance of the magnetite phase on the biotemplate.
The SAED pattern consisted of spots on the rings corresponding
to the diffraction planes (111), (220), (311), (400),
(511) and (440) characteristic of the magnetite phase, which
also revealed the polycrystalline nature of the nanocomposites
(see Figure 3 b). The difference between the polycrystalline
electron diffraction patterns of magnetite and maghemite
phases is solved by the presence of the (111) diffraction
plane, distinctive of the magnetite fcc structure. [24] From the
HR-TEM images (Figure 3 c) we can observe that the obtained
magnetite nanoparticles are single crystalline, as indicated
by the well-resolved lattice fringes. The distance between
two adjacent planes is about 0.255 nm, which corresponds
to the (311) lattice plane in the spinel structure of
Fe 3O 4. [25] The strong peak at 580 cm 1 in the FTIR plot (Figure
3 d) additionally confirms that the phase is magnetite
www.chemeurj.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 7366 – 7373

One-Dimensional Magnetic Nanowires
Figure 3. a) XRD pattern and b) SAED pattern of the fully covered magnetic
nanowires. c) HR-TEM image of a magnetite nanoparticle showing
the interplanar distance; the FFT indicates that the nanoparticle is oriented
in the [001] direction. d) FTIR spectrum of fully covered magnetic
nanowires.
rather than maghemite. The peaks at about 591 and
456 cm 1 in the spectrum indicate the presence of both magnetite
and maghemite. [26]
Magnetic and imaging contrast properties: To evaluate the
potential use of these 1D hybrid magnetic nanostructures
for bioimaging, we first investigated the magnetic properties
of fully magnetite-covered hybrid lysozyme nanofibrils with
the aid of a superconducting quantum interference device
(SQUID), and compared them with those of dispersed magnetic
nanoparticles synthesised under the same solution conditions
in the absence of protein fibrils. Figure 4 shows the
field-dependent magnetisation of the fully covered fibrils
and dispersed magnetic nanoparticles measured at 300 and
5 K, respectively. Their M/H curves at 300 K show nonlin-
FULL PAPER
Figure 4. Magnetisation versus applied magnetic field for: a) dispersed
magnetite nanoparticles, and b) fully covered magnetic nanowires at 5 K
(closed symbols) and 300 K (open symbols). c) Image showing the attraction
of the nanowires to a magnet.
ear, reversible characteristics with no hysteresis (zero coercivity),
features of superparamagnetic behaviour, with saturation
magnetisation (M s) values of about 60 and 47 emu g 1
for fully covered and isolated nanoparticles, respectively.
The observed magnetic behaviour is consistent with the
common understanding that one-dimensionally stacked
magnetic nanoparticles forming a uniform layer behave like
single elongated particles, and so their cooperative response
is more sensitive to a magnetic field. [27] Nevertheless, all the
Chem. Eur. J. 2011, 17, 7366 – 7373 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 7369

above values are lower than the theoretical value of the
bulk magnetite (92 emu g 1 ). [28] A decrease in M is often observed
and is attributed to the surface contribution: spin
canting, surface disorder, stoichiometry deviation, ion distribution
and adsorbed species. [29] The elongated structures of
the nanowires apparently enhance the orientation of the
magnetic moments of the individual nanoparticle constituents,
increasing the net magnetisation. [13, 30] On the other
hand, the magnetisation curves at 5 K show that all samples
have hysteresis with coercitive fields (H c) of 352 and
270 Oe, remnant magnetisation of 16.5 and 10.2 emu g 1 and
M s values of about 90 and 56 emu g 1 for fully covered and
isolated nanoparticles, respectively. The magnetic properties
of the hybrid fibrils were also tested by placing a magnet
near the cuvette. The hybrid nanowires were completely attracted
to the side of the cuvette nearest to the magnet as
displayed in Figure 4 c, which additionally confirms the magnetic
properties of the hybrid samples.
Measurements of magnetisation versus temperature were
performed by standard experimental zero-field-cooling
(ZFC) and field-cooling (FC) protocols with the external
magnetic field set at 500 Oe when necessary. Field-cooled
(FC) magnetisation measurements provide information
about coupling between the magnetic materials because a
FC experiment suppresses thermal fluctuations and increases
net magnetisation. The ZFC and FC magnetisation
curves split below 150 and 195 K, and the ZFC magnetisation
curves exhibit peaks around T = 35 and 105 K for fully
covered fibrils and dispersed Fe 3O 4 nanoparticles, respectively
(see Figure 5), identified as the blocking temperatures
(T B). These data clearly show that there is a temperature
range in which magnetic hybrid fibrils show collective ferromagnetic
behaviour that is unique due to their aggregation
status, as was also the case for nanoparticle chains. [5c,21a]
Nevertheless, superparamagnetism remains after T B as a
consequence of the anisotropic barrier blockage of the magnetisation
orientation of the magnetic layer forming the
hybrid fibril cooled with the ZFC process. [31] The larger T B
and the less steep slope for magnetite nanoparticles in the
above figure could be consistent with: i) a larger dipolar
coupling, [27,32] or ii) larger size and anisotropy in the dispersions
as a result of some degree of aggregation between the
particles, which involves a larger number of nearest neighbouring
particles than in the case of magnetic wires and a
non-regular distribution of the particles in the aggregates, in
contrast to what would be expected in the 1D nanowires.
This implies that dipolar interactions will not equally affect
all particles in the nanoparticle aggregates, giving rise to the
observed broadening in T B.
From the relationship T B=K aV/25k B (in which K a is the
anisotropic constant, and k B the Boltzmann constant), for
uniaxial non-interacting magnetite nanoparticles with a diameter
of 10 nm, T B is found to be 20 K, in contrast to the
values of about 35 K found for the nanowires and 105 K for
dispersed nanoparticles, respectively. From this equation we
can also derive an estimated value for the crystallite size in
the hybrid fibrils and for the dispersed nanoparticles by use
7370
P. Taboada et al.
Figure 5. ZFC-FC plots for: a) dispersed magnetite nanoparticles, and
b) fully covered magnetic nanowires at 5 K (closed symbols) and 300 K
(open symbols). FC curves were obtained in the presence of a magnetic
field of 500 Oe.
of a K a value of 1.35 ”10 4 Jm 3 corresponding to bulk magnetisation.
In this way, the estimated sizes are about 12 and
16 nm, larger than those calculated from XRD data. Possible
reasons for these differences might be the wide size distribution
populations, the magnetic dipole interactions between
nanowires and nanoparticles, the magnetic texture
formed in nanowires due to strong shape anisotropy, the aggregation
state in the dispersed nanoparticles or the disordered
surface spins. These factors can have strong influences
on magnetic properties and, therefore, on calculated sizes.
Additionally, the effective magnetic anisotropy constants of
Fe 3O 4 nanoparticles are normally less than its bulk value
and vary with particle size. [29] All these factors lead to differences
in the estimated particles sizes according to XRD and
magnetic data.
To check the possible efficiency of the biohybrids as novel
T 2 contrast agents, the relaxation times T 1 and T 2* were
measured by NMR dispersion (NMRD) at 9.4 T (400 MHz
proton Larmor frequency, 37 8C). The T 1 (spin–lattice) relaxation
process is the result of the interaction between the excited
nuclei and their surrounding environment, and the T 2*
www.chemeurj.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 7366 – 7373

One-Dimensional Magnetic Nanowires
(spin–spin) relaxation process of the interaction between the
excited nuclei and those with lower energy. The efficiency of
an MRI contrast agent is commonly assessed in terms of its
relaxivities (r 1 and r 2), which are rates of proton relaxation
determined by Equation (1):
1=T i,obs ¼ 1=T i,d þ r i½MŠ ðI ¼ 1, 2Þ ð1Þ
in which 1/T i,obs is the observed solvent relaxation rate in the
presence of a contrast agent, 1/T i,d is the relaxation rate of
the pure diamagnetic solvent, and [M] is the concentration
of the contrast agent. [33] For a T 2 contrast agent, the higher
the r 2*/r 1 ratio the better the contrast efficacy. The spin–spin
and spin–lattice relaxation times weighted spin-echo MRI of
the fully-covered hybrid nanofibrils are shown in Figure 6 a
and Figure S7 in the Supporting Information, respectively.
As shown in the former, the fully covered fibrils exhibited
much stronger enhancement in T 2*-weighted relaxation
times than the dispersed magnetic nanoparticles at the same
Figure 6. a) T 2 relaxation rates as a function of iron concentration (mm
Fe) for fully covered magnetic nanowires obtained by fibril biotemplating;
r 2*=161 mm 1 s 1 .b)T 2*-weighted MR images of phantoms in the
presence of different amounts of magnetic nanowires (expressed in Fe
concentration). 1) 0.1”10 7 , 2) 0.2”10 7 , 3) 0.3”10 7 , 4) 0.5”10 7 ,5)1”
10 7 and 6) 2”10 7 m.
Fe concentration, with an r 2* value of 161 mm 1 s 1 (r 1=
0.42 mm 1 s 1 ) and an r 2*/r 1 ratio of about 390. The effect of
the protein was found to be negligible. The former behaviour
might be a result of the enhanced MR sensitivity due to
clustering of individual magnetic nanocrystals on the fibrils.
[34] Nevertheless, it is necessary to bear in mind that the
large r 2*/r 1 ratio is a result of the different behaviour displayed
by T 1 and T 2 with the applied magnetic field
strength: both T 1 and T 2 generally increase at relatively low
applied magnetic field, but T 1 reaches a maximum around
0.5–1.0 T and then drastically decreases at larger magnetic
fields. In contrast, T 2 remains almost constant or even slightly
increases at larger applied magnetic fields, so the r 2*/r 1
ratio definitely increases with applied magnetic field. [35]
Thus, when comparing r 2*/r 1 ratios it is necessary to bear in
mind the applied magnetic field at which the experiment
was performed. Interestingly, the r 2* relaxivities of the
hybrid fibrils are slightly larger than those obtained for
DNA-templated magnetic nanowires, [11a] (bio)polymer-templated
nanowires, [20,36] magnetic nanoworms [2d, 10b] or conventional
superparamagnetic iron oxide nanoparticles. [37] Such a
strong T 2 shortening effect, typically from stronger magnetic
susceptibility, leads to spin dephasing and substantial MRI
drop, which generated a “darkening” contrast, as seen in the
T 2*-weighted MR images of the hybrids in phantoms measured
at 9.4 T in a preclinical MRI. The mean T 2* values of
the phantoms get lower as the amount of fully covered
nanofibrils increases in solution (seen as black spots in the
images) even though the amount of Fe present is extremely
small, which avoids full darkness of the image (Figure 6 b).
The increased MRI contrast observed for nanowires is the
result of the increase in the susceptibility effects caused by
the presence of Fe that can enhance spin–spin relaxation of
water molecules caused by the slightly larger magnetisation
value [38] and the 1D assembly. [39]
Conclusion
FULL PAPER
We report a method for obtaining magnetic Fe 3O 4 nanowires
based on the use of HSA and Lys protein fibrils as bioscaffolds
for generation of a complete magnetic Fe 3O 4
coating layer on the biotemplate surface by controlled Fe
nanoprecipitation in situ in the presence of NH 4OH. To achieve
full coverage of the protein fibrils a suitable initial [Fe]/ACHTUNG-
TRENUNG[protein] molar ratio must be selected and a suitable
number of sequential additions of Fe ions must be performed.
If these requirements are not fulfilled, partial coverage
and/or protein bundling and further fibril precipitation
are observed. Thanks to their large saturation magnetisation,
the 1-dimensional magnetic nanowires can serve as efficient
MRI contrast agents, with transverse relaxivities
larger than those obtained previously for other 1D nanostructures
derived by different methods. Their capability as
contrast agents was verified by obtaining several MRI
images at different Fe concentrations in phantoms. In spite
of the very low concentrations used, the hybrid nanowires
Chem. Eur. J. 2011, 17, 7366 – 7373 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 7371

exhibit an important contrast imaging effect, which confirms
their potential use as MRI contrast agents. Future work will
involve the refinement of the synthetic process to achieve
perfect control of magnetic coverage, and thus hybrid fibril
widths, magnetic nanowire lengths, population distribution
and in vivo assays.
Experimental Section
Materials: Human serum albumin, lysozyme, FeCl2 and FeCl3 were obtained
from Sigma Chemical. Proteins were used after further purification
by liquid chromatography over a Superdex 75 column equilibrated
with phosphate (0.01 m). All other chemicals were of the highest purity
available. Experiments were carried out with doubly distilled, deionised
and degassed water.
Preparation of HSA and lysozyme fibril solutions: Protein fibril solutions
were prepared by well-established protocols. [16] Briefly, HSA fibril stock
solution was made by dissolving the protein to a final concentration typically
of 2 mg mL 1 in phosphate buffer (pH 7.4, ionic strength 10 mm)
and brought to pH 2.0 by addition of HCl. Lysozyme fibrils were obtained
by forming a stock solution (2 mg mL 1 ) in glycine buffer (pH 2.0,
ionic strength 10 mm). Both protein stock solutions were dialysed extensively
against pure buffer. Protein concentrations were determined spectrophotometrically,
with use of molar absorption coefficients of 35219
and 37 609 m 1 cm 1 at 280 nm for HSA and Lys, respectively. [40] Before incubation,
the solutions were filtered through a 0.2 mm filter into sterile
test tubes. Samples were incubated at 65 8C in a reactor under reflux for
10 and 3 days for HSA and Lys, respectively.
Characterisation of the fibrils: Protein suspensions were applied to
carbon-coated copper grids, blotted, washed, negatively stained with
phosphotungstic acid (2%, w/v), air dried and then examined with a Phillips
CM-12 transmission electron microscope operating at an accelerating
voltage of 120 kV. Samples were diluted 20–200-fold when needed prior
to deposition on the grids.
Magnetic nanowire synthesis: A stock solution (typically 2 g L 1 ) of
either lysozyme or HSA fibrils was diluted 600-fold to avoid the presence
of undesired aggregates during the synthesis of the magnetite nanowire.
Different [Fe]/ACHTUNGTRENUNG[protein] molar ratios (10:1, 25:1 50:1, 100:1, 200:1 and
400:1) and sequential additions (one to five) of the iron salts were tested
to enable the formation of magnetite nanoparticles on the biotemplate
surface and to ensure full coverage of the fibril surface. In a typical synthesis,
an aliquot (100 mL) of lysozyme fibril solution (2 gL 1 , 1.34 ”
10 4 m) was placed in a flask reactor and diluted with doubly distilled, deionised
water (60 mL). Fibril solution was left to incubate at 65 8C for 1 h
with magnetic stirring. Separately, two aqueous solutions containing
FeCl2 (25.4 mg, 0.1 m) and FeCl3, respectively, (64.9 mg, 0.1 m) were prepared
and mixed in a 1:2 molar ratio. After the incubation period at
658C, the temperature of the fibril solution was raised to 80 8C, and a
specific volume of the iron salt mixed solution suitable to get the desired
[Fe]/ACHTUNGTRENUNG[protein] molar ratio was then added under nitrogen. The solution
was left to incubate for 3 h before the addition of NH4OH (1.5 m, 2 mL);
the resulting solution was allowed to react for an additional 1 h and allowed
to cool to room temperature. Stabilisation of the nanowires was
additionally achieved by addition of sodium citrate solution (10 mm,
1 mL) to the resulting nanowires at 80 8C and allowing to incubate for
90 min. The resulting biohybrid solution was dialysed through a dialysis
membrane (SpectraPor, Netherlands, 100 kDa) to remove remaining iron
salts, NH4OH and sodium citrate. If required, fresh additions of the same
volume of Fe salt solutions were carried out under the same conditions
as previously specified to ensure the full magnetic coverage of fibrils and
to create a nanowire.
Characterisation of magnetic 1D assemblies and nanowires: TEM images
were obtained with a transmission electron microscope (Phillips CM-12)
operating at an accelerating voltage of 120 kV as previously described.
HR-TEM images and SAED patterns were obtained with a transmission
7372
electron microscope (Carl-Zeiss Libra 200 FE-EFTEM) operating at
200 kV. X-ray diffraction experiments were carried out with a rotating
anode X-ray generator (Siemens D5005). Twin Gçbel mirrors were used
to produce a well-collimated beam of CuKa radiation (l = 1.5418 A). Xray
diffraction patterns were recorded with an imaging plate detector
(AXS F.Nr. J2–394). FTIR spectra of HSA in aqueous solutions were determined
with a FTIR spectrometer (model IFS-66v from Bruker) with a
horizontal ZnS ATR accessory. The spectra were obtained at a resolution
of 2 cm 1 and generally 200 scans were accumulated to provide a reasonable
signal-to-noise ratio. Magnetic susceptibility measurements were carried
out with a SQUID magnetometer (Quantum Design MPMS5, San
Diego, CA). Iron concentrations of each sample for SQUID, relaxivity
and MRI were determined by inductively coupled plasma atomic emission
spectroscopy (ICP-AES, Varian).
Magnetic resonance measurements and MRI imaging: Transverse and
longitudinal relaxation times were also measured at 9.4 T (400 MHz) and
378C with a Bruker Biospin USR94/20 instrument (Ettlingen, Germany).
An inversion-recovery pulse sequence was used to measure the longitudinal
relaxation times with 16 inversion recovery times logarithmically
spaced. Both Carr–Purcell–Meiboom–Gill (CPMG) and spin-echo pulse
sequences were used to measure transverse relaxation times. MRI phantoms
were constructed in agarose solutions (Sigma–Aldrich, 4%, w/v),
heated and stirred at 80 8C until complete solution of the solid agarose.
Then, with the system still remaining fluid, six HPLC vials (Cromlab,
Spain, 250 mL) were filled with the agarose solution (100 mL) and allowed
to cool to room temperature. Protein solution (5, 10, 15, 25, 50 or
100 mL) was then deposited over the surfaces of the agarose gels, and distilled
water (49, 45, 40, 35, 25 and 0 mL, respectively) was added to the
solutions to make the final volumes even at 150 mL per vial. After a waiting
period of at least 30 min, most of the deposited fluid had been absorbed
by the agarose gels, after which semi-gelificated agarose solution
(50 mL, 10 %, w/v, heated to 80 8C for solution and cooled to about 40–
508C before addition) was added to each phantom to seal them. Imaging
of the phantoms was performed with the previously described Bruker
Biospin system with 440 mT m 1 gradients. A 3D-Gradient Echo image
was acquired (T2*-weighting) with the following parameters: field of
view: 28.8 ”19.2 ”2 mm, matrix size: 288 ” 192 ”40 points giving a spatial
resolution of 100 ”100 ”200 mm, echo time TE = 8 ms, repetition time
TR=100 ms and flip angle = 30.
Acknowledgements
P. Taboada et al.
The authors thank the Ministerio de Ciencia e Innovación (MICINN) for
research project MAT 2010–17336, the Xunta de Galicia for research
project INCITE09206020PR and for European Regional Development
Funds (research project 2010/50), and the Fundación Ramón Areces for
additional financial support. We acknowledge the contributions of B. Argibay,
P. Ramos-Cabrer and J. Castillo from the Clinical Neurosciences
Research Laboratory of the Clinical Hospital of Santiago de Compostela
for the MR images acquired with the 9.4 T MR system.
[1] Y. N. Xia, P. D. Yang, Y. G. Sun, Y. Y. Wu, B. Mayers, B. Gates,
Y. D. Yin, F. Kim, Y. Q. Yan, Adv. Mater. 2003, 15, 353 – 389.
[2] a) C. Ross, Annu. Rev. Mater. Res. 2001, 31, 203 –235; b) J. M. Kinsella,
A. Ivanisevic, J. Phys. Chem. C 2008, 112, 3191 –3193; c) C.
Goubault, P. Jop, M. Fermigier, J. Baudry, E. Bertrand, J. Bibetter,
Phys. Rev. Lett. 2003, 91, 260 802; d) J.-H. Park, G. von Maltzahn, L.
Zhang„ M. P. Schwartz, E. Ruoslathi, S. N. Bathia, M. J. Sailor, Adv.
Mater. 2008, 20, 1630 –1635.
[3] S. Minko, A. Kiriy, G. Gorodyska, M. Stamm, J. Am. Chem. Soc.
2002, 124, 10192.
[4] a) N. Duxin, F. T. Liu, H. Vali, A. Eisenberg, J. Am. Chem. Soc.
2005, 127, 10063 – 10069; b) Y. Piao, J. Kim, H. B. Na, D. Kim, J. S.
Baek, M. K. Ko, J. H. Lee, M. Shokouhimehr, T. Hyeon, Nat. Mater.
2008, 7, 242 –247.
www.chemeurj.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 7366 – 7373

One-Dimensional Magnetic Nanowires
[5] a) V. SalgueiriÇo-Maceira, M. A. Correa-Duarte, M. BaÇobre-
López, M. Grzelczak, M. Farle, L. M. Liz-Marzµn, J. Rivas, Adv.
Funct. Mater. 2008, 18, 616 – 621; b) M. L. Górzny, A. S. Walton,
S. D. Evans, Adv. Funct. Mater. 2010, 20, 1295 –1300; c) K. Nakata,
Y. Hu, O. Uzun, O. Bakr, F. Stellacci, Adv. Mater. 2008, 20, 4294 –
4299; d) S. H. Sun, Adv. Mater. 2006, 18, 393 –403; e) L. Fu, X. G.
Liu, Y. Zhang, V. P. Dravid, C. A. Mirkin, Nano Lett. 2003, 3, 757 –
760; f) S. Palacin, P. C. Hidber, J. P. Bourgoin, C. Miramond, C.
Fermon, G. M. Whitesides, Chem. Mater. 1996, 8, 1316 – 1325.
[6] a) H. Wang, A. J. Patil, S. Petrov, S. Mann, M. A. Winnik, I. Manners,
Adv. Mater. 2009, 21, 1805 – 1808; b) J. Fresnais, J.-F. Berret, B.
Frka-Petesic, O. Sandre, R. Perzynski, Adv. Mater. 2008, 20, 3877 –
3881; c) K. Keren, M. Krueger, R. Girald, G. Ben-Yoseph, U. Sivan,
E. Braun, Science 2002, 297, 72 –75.
[7] a) L. W. Yin, Y. Bando, Y. C. Zhu, D. Goldberg, M. S. Li, Adv.
Mater. 2004, 16, 929 –933; b) J. Sharma, R. Chiabra, A. Cheng, J.
Brownell, Y. Liu, H. Yan, Science 2009, 323, 112 –116; c) T.
Kodama, A. Jain, K. E. Goodson, Nano Lett. 2009, 9, 2005 – 2009.
[8] a) N. C. Bigall, M. Reitzig, W. Naumann, P. Simon, K.-H. Van PØe,
A. Eychmüller, Angew. Chem. 2008, 120, 7994 –7997; Angew. Chem.
Int. Ed. 2008, 47, 7876 –7879; b) S. Banerjee, M. G. C. Kahn, S. S.
Wong, Chem. Eur. J. 2003, 9, 1898 –1908; c) W. Q. Han, A. Zettl,
Nano Lett. 2003, 3, 681 – 683; d) M. B. Dickerson, K. H. Sandhage,
R. R. Naik, Chem. Rev. 2008, 108, 4935 –4978.
[9] S. S. Behrens, J. Mater. Chem. 2008, 18, 3788 –3798.
[10] a) D. Walsh, L. Arcelli, T. Ikoma, J. Tanaka, S. Mann, Nat. Mater.
2003, 2, 386 –390; b) J.-H. Park, G. von Maltzahn, L. Zhang, A. M.
Derfus, D. Simberg, T. J. Harris, E. Ruoslathi, S. N. Bathia, M. J.
Sailor, Small 2009, 5, 694 – 700.
[11] a) S. J. Byrne, S. A. Corr, Y. K. Gun ko, J. M. Kelly, D. F. Brougham,
S. Ghosh, Chem. Commun. 2004, 2560 – 2561; b) J. M. Kinsella, A.
Ivanisevic, Langmuir 2007, 23, 3886 – 3890; c) Q. Gu, D. T. Haynie,
Mater. Lett. 2008, 62, 3047 – 3050.
[12] a) M. Allen, J. W. M. Bulte, L. Liepold, G. Basu, H. A. Zywicke,
J. A. Frank, M. Young, T. Douglas, Magn. Reson. Med. 2005, 54,
807 – 812; b) A. A. Martinez-Morales, N. G. Portney, Y. Zhang, G.
Destito, G. Budak, E. Ozbay, M. Manchester, C. S. Ozkan, M.
Ozkan, Adv. Mater. 2008, 20, 4816 –4820.
[13] I. A. Banerjee, L. Yu, M. Shima, T. Yoshino, H. Takeyama, T. Matsunaga,
H. Matsui, Adv. Mater. 2005, 17, 1128 – 1131.
[14] a) R. Tsukamoto, M. Muraoka, M. Seki, H. Tabata, I. Yamashita,
Chem. Mater. 2007, 19, 2389 –2391; b) M. Knez, A. M. Bittner, F.
Boes, C. wege, H. Jeske, E. Maiß, K. Kern, Nano Lett. 2003, 3,
1079 – 1082.
[15] a) C. M. Dobson, Nature 2003, 426, 884 – 890; b) I. Cherny, E. Gazit,
Angew. Chem. 2008, 120, 4128 –4136; Angew. Chem. Int. Ed. 2008,
47, 4062 –4069; c) T. R. Jahn, S. E. Radford, Arch. Biochem. Biophys.
2008, 469, 100 –117.
[16] a) P. Taboada, S. Barbosa, E. Castro, V. Mosquera, J. Phys. Chem. B
2006, 110, 20733 –20736; b) J. Juµrez, P. Taboada, V. Mosquera, Biophys.
J.2009, 96, 2353 –2370; c) E. Frare, P. P. De Laureto, J. Zurdo,
C. M. Dobson, A. Fontana, J. Mol. Biol. 2004, 340, 1153 –1165;
d) A. P. Pawar, K. F. Dubai, J. Zurdo, F. Chiti, M. Vendruscolo,
C. M. Dobson, J. Mol. Biol. 2005, 350, 379 –392; e) V. H. Lieu, J. W.
Wu, S. S. S. Wang, C. H. Wu, Biotechnol. Prog. 2007, 23, 698 –706.
[17] T. Peters, Jr., All About Albumin: Biochemistry Genetics, and Medical
Applications, Academic Press, New York, 1996.
[18] a) W. J. Crookes-Goodson, J. M. Slocik, R. N. Naik, Chem. Soc. Rev.
2008, 37, 2403 –2412; b) N. Ostrov, E. Gazit, Angew. Chem. 2010,
FULL PAPER
122, 3082 – 3085; Angew. Chem. Int. Ed. 2010, 49, 3018 –3021; c) M.
Malisauskas, R. Meskys, L. A. Morozova-Roche, Biotechnol. Prog.
2008, 24, 1166 –1170.
[19] L. Yu, I. A. Banerjee, M. Shima, K. Rajan, H. Matsui, Adv. Mater.
2004, 16, 709 –712.
[20] S. R. Bull, M. O. Guller, R. A. Bras, T. J. Meade, S. I. Stupp, Nano
Lett. 2005, 5, 1–4.
[21] S. Ghosh, A. Mukherjee, P. J. Sadler, S. Verma, Angew. Chem. 2008,
120, 2249 –2253; Angew. Chem. Int. Ed. 2008, 47, 2217 – 2221.
[22] Y. Sahoo, A. Goodarzi, M. T. Swihart, T. Y. Ohulchanskyy, N. Kaur,
E. P. Furlani, P. N. Prasad, J. Phys. Chem. B 2005, 109, 3879 – 3885.
[23] P. Dutta, A. Manivannan, M. S. Seehra, N. Shah, G. P. Huffman,
Phys. Rev. B 2004, 70, 174 428.
[24] I. Martínez-Mera, M. E. Espinosa-Pesqueira, R. PØrez-Hernµndez, J.
Arenas-Alatorre, Mater. Letters. 2007, 61, 4447 –4451.
[25] J. Wan, W. Cai, J. Feng, X. Meng, E. Liu, J. Mater. Chem. 2007, 17,
1188 – 1192.
[26] a) H. Wang, Q.-W. Chen, L.-X. Sun, H.-p. Qi, X. Yang, S. Zhou, J.
Xiong, Langmuir 2009, 25, 7135 – 7139; b) T. J. Daou, G. Pourroy, S.
BØgin-Colin, J. M. Gren›che, C. Ulhaq-Bouillet, P.- LegarØ, P. Bernhardt,
C. Leuvrey, G. Rogez, Chem. Mater. 2006, 18, 4399 –43404.
[27] a) A. F. Gross, M. R. Diehl, K. C. Beverly, E. K. Richman, S. H. Tolbert,
J. Phys. Chem. B 2003, 107, 5475 –5482; b) D. Baldomir, D. Serantes,
M. Pereiro, J. Botana, J. E. Arias, S. H. Masunaga, J. Rivas, J.
Nanosci. Nanotechnol. 2010, 10, 2717 –2721.
[28] D. H. Han, H. L. Wang, J. Luo, J. Magn. Magn. Mater. 1994, 136,
176 – 182.
[29] G. F. Goya, T. S. Berquó, F. C. Fonseca, M. P. Morales, J. Appl. Phys.
2003, 94, 3520 –3528.
[30] J. Qin, S. Laurent, Y. S. Jo, A. Roch, M. Mikhaylova, Z. M. Bhujwalla,
R. N. Muller, M. Muhammed, Adv. Mater. 2007, 19, 1874 –1878.
[31] Y. Wang, Y. W. Ng, Y. Chen, B. Shuter, J. Yi, J. Ding, S.-c. Wang, S.-
S. Feng, Adv. Funct. Mater. 2008, 18, 308 – 318.
[32] L. Zhang, Y. Zhang, J. Magn. Magn. Mater. 2009, 321, L15 – L20.
[33] N. Bloembergen, E. M. Purcell, R. V. Pound, Phys. Rev. 1948, 73,
679 – 712.
[34] J. F. Berret, N. Schonbeck, F. Gazeau, D. El Kharrat, O. Sandre, A.
Vacher, M. Airiau, J. Am. Chem. Soc. 2006, 128, 1755 –1761.
[35] P. Caravan, C. T. Farrar, L. Frullano, R. Uppal, Contrast Media Mol.
Imaging 2009, 4, 89–100.
[36] a) S. A. Corr, S. J. Byrne, R. Tekoriute, C. J. Meledandri, D. F. Bougham,
M. Lynch, C. Kerskens, L. O Dwyer, Y. K. Gun ko, J. Am.
Chem. Soc. 2008, 130, 4214 – 4215; b) M. Y. Shin, S. I. Kim, S. J. Lee,
K. E. Lee, S.-S. Han, D.-P. Jang, Y.-B. Kim, Z.-H. Cho, I. So, G. M.
Spinks, Langmuir 2008, 24, 12 107–12 111.
[37] H. B. Na, I. C. Song, T. Hyeon, Adv. Mater. 2009, 21, 2133 –2148.
[38] J. H. Lee, Y. M. Huh, Y. Jun, J. Seo, J. Jang, H. T. Song, S. Kim, E. J.
Cho, H. G. Yoon, J. S. Suh, J. Cheon, Nat. Med. 2006, 13, 95–99.
[39] J. M. Perez, L. Josephson, T. O Loughlin, D. Hogemann, R. Weissleder,
Nat. Biotechnol. 2002, 20, 816 –820.
[40] a) C. N. Pace, F. Vajdos, L. Fee, G. Grimsley, T. Gray, Protein Sci.
1995, 4, 2411 –2423; b) T. Knubovets, J. J. Osterhout, P. J. Connolly,
A. M. Klibanov, Proc. Natl. Acad. Sci. USA 1999, 96, 1262 – 1267.
Received: December 20, 2010
Revised: March 7, 2011
Published online: May 6, 2011
Chem. Eur. J. 2011, 17, 7366 – 7373 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 7373

6.7.- Relevant aspects on the HSA fibril formation
I. HSA has the ability to self-assemble into amyloid-like aggregates under different
solution conditions, i.e., under both physiological and acidic pH at elevated
temperature and different ionic strengths. Under these solution conditions, the HSA
native state is destabilized generating partially folded states that can aggregate to
form fibrils.
II. At physiological pH, fibrillation is progressively faster and more efficient in the
presence of up to 50 mM NaCl due to electrostatic shielding. ThT fluorescence, CD, FT-
IR and Trp fluorescence spectra confirm the structural changes in both tertiary and
secondary structure along the HSA fibrillation process. In this way, large extents of β-
sheet structure at large salt concentrations are also corroborated from the analysis of
far UV-CD spectra.
III. Under acidic conditions, a progressive enhancement of HSA fibrillation is observed as
electrolyte concentration in solution increases.
IV. The fibrillation process of HSA does not show a lag-phase growth, except at acidic pH
in the absence of NaCl. The HSA fibrillation is a downhill process which does not
require a highly organized and unstable nucleus, with a progressive increase of the β-
sheet (up to 26%) and an unordered conformation at the expense of the α-helical
conformation.
V. The fibrils obtained show a curly morphology and differ in length. Besides,
suprafibrillar assemblies (spherulites and fibrillar gels) formed by the protein human
serum albumin under different solution conditions.
VI. Upon incubation at 65 0 C at both acidic (pH 2.5) and physiological (pH 7.4) pH in the
presence of different amounts of added electrolyte, suprafibrillar assemblies,
spherulites and fibrillar gels, are formed under different solution conditions. Fibrillar
gels are formed through intermolecular nonspecific association of amyloid fibrils.
Meanwhile, at a pH close to the isoelectric point of HSA (pH 5.5), particulate gels were
observed as a consequence of a faster protein aggregation, which does not allow the
necessary structural reorganization to enable the formation of more ordered
structures.
VII. Within spherulites, fibrils display a radial arrangement around a disorganized protein
core with sizes of several micrometers, as revealed by POM and confocal microscopy.
VIII. Protein structure destabilization and subsequent aggregation (into amorphous or well-
defined structured aggregates) were conditioned by the experimental pH,
215

temperatures, and solvent polarity.
IX. Ethanol regulated the hydrogen bonding extension, the attractive hydrophobic
interactions, and the protein accessible surface area which, in turn, give rise to
intermediate structural states prone to form aggregate.
X. The HSA aggregation rate at physiological condition is faster than under acidic one
owing to the pre-existing structural differences of the protein molecules in solution
and to changes in the nature and strength of intermolecular interaction upon
incubation.
XI. Ethanol promoted an α-helix to β-sheet conformational transition at intermediate
alcohol concentrations, whereas at low and high ethanol contents α-helix prevailed as
the predominant structure at room temperature and physiological pH.
XII. Electrostatic and hydrophobic interactions are important to control stacking of β-
sheets favouring the formation of amyloid-like fibrils at physiological pH and high
temperature.
6.8.- Relevant aspects on the gold and magnetic nanowires
I. We report a method to obtain metallic Au nanowires by using Lys protein fibrils as
bioscaffolds. The metallic coverage of nanofibrils could be easily controlled by
changing the numbers of sequential additions of gold growth solution into the fibril
solution overall already preformed Au nanocrystals. In this way, a continuous coverage
of the fibril surface is obtained as a result of the growth of pre-existing nanoparticles
and generation of new ones on the fibrils surface and subsequent fusion of adjacent
as-synthesized nanoparticles.
II. UV-Vis spectra results showed that the hybrid fibrils can successfully catalyze the
reduction reaction from p-nitrophenol to p-aminophenol by NaBH4. Moreover, the
fibril metallic fibrils provide larger reaction rates values 1.5-4 than other hybrid
materials reported, previously, as for example glucose-reduced Au nanoparticle
networks, PAMAM-dendrimer-supported spherical nanoparticles amongst others.
III. On the other hand, we report a method for obtaining magnetic Fe3O4 nanowires based
on the use of HSA and Lys protein fibrils as bioscaffolds for generation of a complete
magnetic coating layer on the biotemplate surface by controlled Fe nanoprecipitation
in situ in the presence of NH4OH.
IV. Thanks to their large saturation magnetization, the 1D magnetic nanowires can serve
216

as efficient MRI contrast agents, with transverse relaxivities larger than those obtained
previously for other 1D nanostructures derived from different methods. Their
capability as contrast agents was verified by obtaining MRI images at different Fe
concentrations in phantoms.
217

Conclusions
The work has two parts: The first one was focused on the characterization of three block
copolymers (EO12SO10, EO10SO10EO10, and EO137SO18EO137), meanwhile the second part was
dedicated to the study of the fibrillation of the protein human serum albumin that was cover
with gold and magnetic nanoparticles.
The general conclusions are:
1. - Characterization of the block copolymers EO12SO10, EO10SO10EO10, y EO137SO18EO137, in
solution at the air/water and chloroform/water interfaces.
I. For E12S10 dilute solutions, the coexistence of spherical micelles with vesicular
structures has been observed. In addition, the spontaneous formation of vesicles by
poly(oxystyrene)-poly(oxyethylene) block copolymers is reported for the first time, as
confirmed by light scattering, polarized light microscopy, and transmission and cryo-
scanning electron microscopy data.
II. In dilute solution, the self-assembly of copolymer E10S10E10 leads to the formation of
elongated micelles as supported by light scattering and transmission electron
techniques.
III. In the case of copolymer E137S18E137, typical spherical micelles are observed. Tube
inversion and rheological measurements were used to define the sol- soft- and gel-
hard boundaries of this copolymer.
IV. E12S10 and E10S10E10 copolymers did not form gels in the concentration range analysed.
However, only certain concentrations of copolymer E10S10E10 were analysed by
rheometry. From experimental data, an upturn in the low-frequency range of the
stress moduli was observed, denoting the existence of an emerging slow process,
which was assigned to the formation of an elastic network.
V. Block copolymers E12S10, E10S10E10 and E137S18E137 showed spontaneous adsorption at
the air-water interface, which slowed down when the hydrophobicity of the molecule
was increased. In contrast, at the chloroform/water interface no measurable effect is
observed for copolymer E12S10 due to the slow diffusion of its chains to the interface in
219

combination with a low bulk concentration; this slow diffusion is associated with the
fact that chloroform is a good solvent for both E and S blocks.
VI. Copolymer E137S18E137 displays an adsorption isotherm with the four classical regions
representing the pancake, mushroom, brush and condensed states; the presence of a
pseudo-plateau is attributed to the pancake-to-brush transition as E chains submerge
into the aqueous sub-phase. On the other hand, for E12S10 and E10S10E10 copolymers
only two regions are observed in their adsorption isotherms as a consequence of their
low molecular weights, short S and E block lengths, and much larger S/E ratio. This
involves the disappearance of the pseudo-plateau region due to the decrease in the
fractional interfacial area occupied by EO segments.
VII. According to AFM images, circular micelles are observed on Langmuir-Blodgett films of
the copolymers obtained at two surface transfer pressures. A decrease in micelle size
and an increase in monolayer thickness are observed with increases in transfer
pressure. Aggregation numbers derived from AFM images increase with the increase
of the S weight fraction at a certain deposition pressure, which can be a consequence
of stronger attractive interactions between the S blocks to avoid contact with the
solvent.
2. - On the HSA fibrillation:
I. HSA has the ability to self-assemble into amyloid-like aggregates under different
solution conditions, i.e., under both physiological and acidic pH at elevated
temperature and different ionic strengths and solvent compositions. Under these
solution conditions, the HSA native state is destabilized generating partially folded
states that can aggregate to form fibrils.
II. At physiological pH, fibrillation is progressively faster and more efficient in the
presence of up to 50 mM NaCl due to electrostatic shielding. ThT fluorescence, CD, FT-
IR and Trp fluorescence spectra confirm the structural changes in both tertiary and
secondary structure along the HSA fibrillation process. In this way, large extents of β-
sheet structure at large salt concentrations are also corroborated from the analysis of
far UV-CD spectra.
III. Under acidic conditions, a progressive enhancement of HSA fibrillation is observed as
electrolyte concentration in solution increases.
IV. The fibrillation process of HSA does not show a lag-phase growth, except at acidic pH
in the absence of NaCl. The HSA fibrillation is a downhill process which does not
220

equire a highly organized and unstable nucleus, with a progressive increase of the β-
sheet (up to 26%) and an unordered conformation at the expense of the α-helical
conformation.
V. The fibrils obtained show a curly morphology and differ in length. Besides,
suprafibrillar assemblies (spherulites and fibrillar gels) formed by the protein human
serum albumin under different solution conditions.
VI. Upon incubation at 65 0 C at both acidic (pH 2.5) and physiological (pH 7.4) pH in the
presence of different amounts of added electrolyte, suprafibrillar assemblies,
spherulites and fibrillar gels, are formed under different solution conditions. Fibrillar
gels are formed through intermolecular nonspecific association of amyloid fibrils.
Meanwhile, at a pH close to the isoelectric point of HSA (pH 5.5), particulate gels were
observed as a consequence of a faster protein aggregation, which does not allow the
necessary structural reorganization to enable the formation of more ordered
structures.
VII. Within spherulites, fibrils display a radial arrangement around a disorganized protein
core with sizes of several micrometers, as revealed by POM and confocal microscopy.
VIII. Protein structure destabilization and subsequent aggregation (into amorphous or well-
defined structured aggregates) were conditioned by the experimental pH,
temperatures, and solvent polarity.
IX. Ethanol regulated the hydrogen bonding extension, the attractive hydrophobic
interactions, and the protein accessible surface area which, in turn, give rise to
intermediate structural states prone to form aggregate.
X. The HSA aggregation rate at physiological condition is faster than under acidic one
owing to the pre-existing structural differences of the protein molecules in solution
and to changes in the nature and strength of intermolecular interaction upon
incubation.
XI. Ethanol promoted an α-helix to β-sheet conformational transition at intermediate
alcohol concentrations, whereas at low and high ethanol contents α-helix prevailed as
the predominant structure at room temperature and physiological pH.
XII. Electrostatic and hydrophobic interactions are important to control stacking of β-
sheets favouring the formation of amyloid-like fibrils at physiological pH and high
temperature.
3. - On the metal and magnetic nanowires :
I. We report a method to obtain metallic Au nanowires by using Lys protein fibrils as
221

ioscaffolds. The metallic coverage of nanofibrils could be easily controlled by
changing the numbers of sequential additions of gold growth solution into the fibril
solution overall already preformed Au nanocrystals. In this way, a continuous coverage
of the fibril surface is obtained as a result of the growth of pre-existing nanoparticles
and generation of new ones on the fibrils surface and subsequent fusion of adjacent
as-synthesized nanoparticles.
II. UV-Vis spectra results showed that the hybrid fibrils can successfully catalyze the
reduction reaction from p-nitrophenol to p-aminophenol by NaBH4. Moreover, the
fibril metallic fibrils provide larger reaction rates values 1.5-4 than other hybrid
materials reported, previously, as for example glucose-reduced Au nanoparticle
networks, PAMAM-dendrimer-supported spherical nanoparticles amongst others.
III. On the other hand, we report a method for obtaining magnetic Fe3O4 nanowires based
on the use of HSA and Lys protein fibrils as bioscaffolds for generation of a complete
magnetic coating layer on the biotemplate surface by controlled Fe nanoprecipitation
in situ in the presence of NH4OH.
IV. Thanks to their large saturation magnetization, the 1D magnetic nanowires can serve
as efficient MRI contrast agents, with transverse relaxivities larger than those obtained
previously for other 1D nanostructures derived from different methods. Their
capability as contrast agents was verified by obtaining MRI images at different Fe
concentrations in phantoms.
222

References
1. Hiemenz, Paul C. Polymer Chemistry: The basic consepts. New York: Marcel Dekker, Inc.,
1984. 0-8247-7082-X.
2. Ebewele, R. O. Polymer science and technology. New York: CRC Press, 2000.
3. Sun, S. F. Physical Chemistry of Macromolecules: Basic principles and issues. New York: John
Wiley & Sons, Inc., 2004. 0-471-28138-7.
4. Berg, J. M.; Tymoczko, J. L.; Stryer, L.; Clarke, N. D. Biquímica. Barcelona: Editorial Reverté,
S. A., 2003.
5. Vicent, María C.; Álvarez, Silvia; Zaragozá, José L. Ciencia y tecología de Polímeros.
Valencia : Universdiad Politécnica de Valencia, 2006.
6. Beyond molecules: Self-assembly of mesoscopic and macroscopic components. Whitesides,
G. M.; Boncheva, M. 2002, PNAS, Vol. 99, pp. 4769-4774.
7. Rao, C. N. R.; Müller, A.; Cheetham, A. K. Nanomaterials chemistry: recent developments
and new directions. Weinheim : WILEY-VCH, 2007.
8. Self-assembling materials for therapeutic delivery. Branco, M. C.; Schneider, J. P. 2009, Acta
Biomaterialia, Vol. 5, pp. 817-831.
9. Directing the self-assembly of block copolymers. Darling, S. B. 2007, Prog. Pol. Sci., Vol. 32,
pp. 1152-1204.
10. Solution self-assembly of tailor-made macromolecular building blocks prepared by
controlled radical polymerization techniques. Lutz, Jean F. 2006, Polym Int, Vol. 55, pp. 979-
993.
11. Self-Assembly at All Scales. Whitesides, George M.; Grzybowski, Bartosz. 2002, Science,
Vol. 295, pp. 2418-2421.
12. Rotello, V. M.; Thayumanavan, S. Molecular recognitionand polymers: Control of polymer
structure and self-assembly. Hoboken, New Jersey: John Wiley & Sons, Inc., 2008.
13. Bottom-up Nanoelectronics. Hadley, Peter. Amsterdam: 34 European Microwave
Conference, 2004, pp. 141-145.
14. Technologies for nanofluidic systems: top-down vs. bottom-up—a review. Mijatovic, D.;
Eijkel, J. C. T.; van den Berg, A. 2005, Lab Chip 5, Vol. 5, pp. 492-500.
15. Hamley, I. W. Block copolymers in solutions: fundamentals and application. John Wiley &
Sons, Ltd, 2005.
223

16. Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry. New York:
Marcel Dekker, 1997.
17. Hamley, I. W. Introduction to soft matter: synthetic and biological self-assembling
materials. Chichester: John Wiley & Sons, Ltd, 2007.
18. Petty, M. C. Langmuir-blodgett films: an introduction. Cambridge: Cambridge University
Press, 1996.
19. Adamson, A. W.; Gast, A. P. Physical chemistry of surfaces. 6th ed. New York: John Wiley &
Sons, Inc., 1997.
20. Dynamic surface tension and dilational viscoelasticity of adsorption layers of a
hydrophobically modified chitosan. Babak, Valery G.; Desbrièresb, Jacques; Tikhonova,
Vladimir E. 2005, Colloids and Surfaces A: Physicochem. Eng. Aspects, Vol. 255, pp. 119-130.
21. Adsorption kinetics of hydrophobic polysoaps at the methylene chloride–water interface.
Babak, Valery; Boury, Frank. 2004, Colloids and Surfaces A: Physicochem. Eng. Aspects, Vol.
243, pp. 33-42.
22. Dilational viscoelasticity and relaxation properties of interfacial electrostatic complexes
between oppositely charged hydrophobic and hydrophilic polyelectrolytes. Babak, V. G.;
Barosb, F.; Desbrièresd, J. 2008, Colloids and Surfaces B: Biointerfaces, Vol. 65, pp. 43-49.
23. Schärtl, Wolfgang. Light Scattering from Polymer Solutions and Nanoparticles Dispersions.
Berlin: Springer, 2007.
24. Berne, B. J.; Pecora, R. DYNAMIC LIGHT SCATTERING: with application to chemistry,
biology, and physics. New York: John Wiley & Sons, INC., 2000.
25. Xu, R. Particle characterization: light scattering methods. New York: Kluwer Academic
Publishers, 2002.
26. Teraoka, I. Polymer Solutions: An Introduction to Physical Properties. Iwao Teraoka.
Brookling: John Wiley & Sons, Inc, 2002.
27. Some applications of light scattering in materials science. Holoubek, J. 2007, Journal of
Quantitative Spectroscopy & Radiative Transfer, Vol. 106, pp. 104-121.
28. Uchegbu, I. F.; Schätzlein, A. G. Polymer in drug delivery. Boca Raton: Taylor & Francis,
2006.
29. Young, R. J.; Lovell, P. A. Introduction to polymers. London: Chapman & Hall, 1991.
30. Huglins, M. B. Ligth scattering from polymer solution. New York: Plenum Press, 1972.
31. Kokhanovsky, A. A. Light Scattering Reviews 4: single light scattering and radiative
transfer. Chichester: Praxis Publishing, 2009.
32. Static and dynamic light scattering of biological macromolecules: what can we learn?
Murphy, R. M. 1997, Current Opinion in Biotechnology, Vol. 8, pp. 25-30.
224

33. Wang, Z. L. HANDBOOK OF MICROSCOPY FOR NANOTECHNOLOGY. Boston: KLUWER
ACADEMIC PUBLISHERS, 2005.
34. Electron Diffraction Using Transmission Electron Microscopy. Bendersky, L. A.; Gayle, F. W.
2001, J. Res. Natl. Inst. Stand. Technol., Vol. 106, pp. 997-1012.
35. van Dyck, S. A.; van Landuyt, D. J.; van Tendeloo, G. Electron Microscopy: Principles and
Fundamentals. VCH, 1997.
36. Yao, N.; Wang, Z. L. HANDBOOK OF MICROSCOPY FOR NANOTECHNOLOGY. BOSTON:
KLUWER ACADEMIC PUBLISHERS, 2005.
37. Goldstein, J. I.; Newbury, D. E.; Echlin, P.; Joy, D. C.; Lyman, C. E.; Lifshin, E.; Sawyer, L.;
Michael, J. R. Scanning Electron Microscopy and X-Ray Microanalysis. New York: Kluwer
Academic/Plenum Publishers, 2003.
38. Bowen, W. R.; Hilal, N. Atomic force microscopy in process engineering: an introduction to
AFM for improved processes and products. Elsevier, 2009. pp. 1-24. Vol. 1.
39. Valeur, B. Molecular fluorescence principles and application. Weinheim: Wiley-VCH, 2002.
40. van Holde, K. E.; Johnson, W. C.; Ho, P. S. Principles of Physical Biochemistry. Upper Saddle
River: Pearson Prentice-Hall, 2006.
41. The binding of thioflavin-T to amyloid fibrils: localisation and implications. Krebs, M. R. H.;
Bromley, E. H. C.; Donald, A. M. 2005, Journal of Structural Biology, Vol. 149, pp. 30-37.
42. Wallace, B. A.; Janes, R. W. Modern techniques for circular dichroism and synchrotron
radiation ciicular dichroism spectroscopy. Amsterdam: IOS Press, 2009.
43. A Holistic Approach for Protein Secondary Structure Estimation from Infrared Spectra in
H2O Solutions. Vedantham, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. 2000,
Analytical Biochemistry, Vol. 285, pp. 33-49.
44. Schrander, B. Infrered and raman spectroscopy: methods and applications. Weinheim:
VCH, 1995.
45. Protein secondary structures in water from second-derivative amide I infrared spectra.
Dong, A.; Huang, P.; Caughey, W. S. 1990, Biochemistry, Vol. 29, pp. 3303-3308.
46. Secondary structures of proteins adsorbed onto aluminum hydroxide: Infrared spectroscopic
analysis of proteins from low solution concentrations. Dong, A.; Jones, L. S.; Kerwin, B. A.;
Krishnan, S.; Carpenter, J. F. 2006, Analytical Biochemistry, Vol. 351, pp. 282-289.
47. Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy. Bagínska,
K.; Makowska, J.; Wiczk, W.; Kasprzykowski, F.; Chmurzynski, L. 2008, J. Pept. Sci., Vol. 14, pp.
283-289.
48. Hunt, B. J.; James, M. I. polymer Characterisation. New York: Marcel Dekker, 1993.
225

49. Goodwin, J. W.; Hughes, R. W. Rheology for chemists: an introduction. Cambridge: RSC
Plublishing, 2008.
50. Guinebretière, R. X-ray Diffraction by Polycrystalline Materials. London: ISTE Ltd, 2007.
51. He, B. B. Two-dimensional X-Ray diffraction. New Jersey: John Wiley & Sons, 2009.
52. Sun, S. F. Physical Chemistry of Macromolecules: Basic principles and issues. John Wiley &
Sons, Inc., 2004. 0-471-28138-7.
53. He, B. B. Two-dimensional X-Ray diffraction. New Jersey : John Wiley & Sons, 2009.
54. Buchner, J.; Kiefhaber, T. Protein folding handbook. Weinheim : WILEY-VCH, 2005.
55. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of instrumental analysis. Canada:
Thomson Brooks/Cole, 2007.
56. Murphy, D. P. Fundamentals of light microscopy and electronic imaging. Sumerset: WILEY-
LISS, 2001.
57. A Novel Bottom-Viewed Inductively Coupled Plasma-Atomic Emission Spectrometry. Chan,
G. C. Y.; Chan, W. T. 2004, Spectrochimica Acta Part B, Vol. 59, pp. 41-58.
58. Clarke, J.; Braginski, A. I. The SQUID handbook. Weinheim : WILEY-VCH, 2004. Vol. II
Applications of SQUIDs and SQUID systems.
59. Magnetic Nanoparticles for Drug Delivery. Arruebo, M.; Fernández-Pacheco, R.; Ibarra, M.
R.; Santamaría, J. 2007, Nanotoday, Vol. 2, pp. 22-32.
60. Mørup, S.; Hansen, M. F. Superparamagnetic Particles. H. Kronmüller and S. Parkin.
Handbook of Magnetism and Advanced Magnetic Materials. John Wiley & Sons, 2007, Vol. 4.
61. Andrä, W.; Häfeli, U.; Hergt, R.; Misri, R. Application of Magnetic Particles in Medicine and
Biology. [book auth.] H. Kronmüller and S. Parkin. Handbook of Magnetism and Advanced
Magnetic Materials. John Wiley & Sons, 2007, Vol. 4.
62. Nikiforov, V. N.; Filinova, E. Y. Biomedical Applications of Magnetic Nanoparticles. [book
auth.] S. P. Gubin. Magnetic Nanoparticles. Weinheim: WILEY-VCH, 2009, Vol. 10.
63. Goyen, M. Real whole-body MRI: requirements, indications, perspectives. New York: The
McGraw-Hill Companies, Inc, 2008.
64. Stroman, P. W. Essentials of functional MRI. Boca Raton, London, New York : CRC Press,
2011.
65. Lecommandoux, SébastienLazzari, Massimo; Liu, Guojun. An Introduction to Block
Copolymer Applications: State-of-the-Art and Future Developments. Massimo Lazzari, Guojun
Liu and Sébastien Lecommandoux. Block Copolymers in Nanoscience. Germany: WILEY-VCH,
2006.
66. Hamley, Ian W. The Physics of Block Copolymers. Oxford: Oxford University Press, 1998.
226

67. Polymers Get Organized. Bucknall , David G.; Anderson, Harry L. 2003, Science, Vol. 302,
pp. 1904-1905.
68. Aggregation of water-soluble block copolymers in aqueous solutions: Recent trends.
Nakashima, Kenichi; Bahadur, Pratap. 2006, Advances in Colloid and Interface Science , Vols.
123-126, pp. 75–96.
69. Polydispersity and block copolymer self-assembly. Lynda, Nathaniel A.; Meulerb, Adam J.;
Hillmyer, Marc A. 2008, Progress in Polymer Science, Vol. 33, pp. 875-893.
70. Ozin, Geoffrey A.; Arsenault, André C. Self-Assemblig Block Copolymers. Geoffrey A. Ozin
and André C. Arsenault. Nanochemistry: A chemical approach to nanomaterials. Toronto: RSC
Publishing, 2005.
71. Structural Properties of Self-assembled Polymeric Aggregates in Aqueous Solutions.
Mortensen, Kell. 2001, Polym. Adv. Technol. , Vol. 12, pp. 2-22.
72. Self-Assembly of Block Copolymer Micelles in an Ionic Liquid. He, Y.; Li, Z.; Simone, P.;
Lodge, T. P. 2006, J. Am. Chem. Soc., Vol. 128, pp. 2745-2750.
73. Hubbard, A. T. Encyclopedia of Surface and Colloid Science. New York: Marcel Dekker, Inc.,
2002.
74. Interaction of polymerizing systems with rubber and its homologs. Part II. Interaction of
rubber in the polymerization of methyl methacrylate and of styrene. Merrett, F. M. 1954,
Trans. Faraday Soc., Vol. 50, pp. 759-767.
75. A Supramolecularly Assisted Transformation of Block-Copolymer Micelles into Nanotubes.
Kim, Sung H.; Nederberg, Frederick; Jakobs, Robert; Tan, Jeremy P. K.; Fukushima, Kazuki;
Nelson, Alshakim ; Meijer, E. W.; Yang, Yi Y.;. 2009, Angew. Chem. Int. Ed., Vol. 48, pp. 4508-
4512.
76. Self-Assembled Block Copolymer Aggregates: From Micelles to Vesicles and their Biological
Applications. Blanazs, A.; Armes, S. P.; Ryan, A. J. 2009, Macromol. Rapid Commun., Vol. 30,
pp. 267-277.
77. Structural study on the micelle formation of poly(ethylene oxide)-poly(propylene oxide)poly(ethylene
oxide) triblock copolymer in aqueous solution. Mortensen, K.; Pedersen, J. S.
1993, Macromolecules, Vol. 26, pp. 805-812.
78. Structure of (Deuterated PEO)−(PPO)−(Deuterated PEO) Block Copolymer Micelles As
Determined by Small Angle Neutron Scattering. Goldmints, I.; Yu, G. E.; Booth, C.; Smith, K. A.;
Hatton, T. A. 1999, Langmuir, Vol. 15, pp. 1651-1656.
79. Small-Angle Neutron Scattering Investigation of the Temperature-Dependent Aggregation
Behavior of the Block Copolymer Pluronic L64 in Aqueous Solution. Yang, L.; Alexandridis, P.;
Steyler, D. C.; Kositzia, M. J.; Holwarth, J. F. 2000, Langmuir, Vol. 16, pp. 8555-8561.
80. A Supramolecularly Assisted Transformation of Block-Copolymer Micelles into Nanotubes.
Kim, Sung H.; Nederberg, Frederick; Jakobs, Robert; Tan, Jeremy P. K.; Fukushima, Kazuki;
227

Nelson, Alshakim ; Meijer, E. W.; Yang, Yi Y. 2009, Angew. Chem. Int. Ed., Vol. 48, pp. 4508-
4512.
81. Influence of Ionic Surfactants on the Aggregation of Poly(Ethylene Oxide)-Poly(Propylene
Oxide)-Poly(Ethylene Oxide) Block Copolymers Studied by Differential Scanning and Isothermal
Titration Calorimetry . da Silva, R. C.; Olofsson, G.; Schillén, K.; Loh, W. 2002, J. Phys. Chem. B,
Vol. 106, pp. 1239-1246.
82. Association and Surface Properties of Poly(ethylene oxide)−Poly(styrene oxide) Diblock
Copolymers in Aqueous Solution. Mai, S. M.; Booth, C.; Kelaraskis, A.; Haverdraki, V.; Ryan, A.
J. 2000, Langmuir, Vol. 16, pp. 1681-1688.
83. Association Properties of a Diblock Copolymer of Ethylene Oxide and Styrene Oxide in
Aqueous Solution Studied by Light Scattering and Rheometry. Kelaraski, A.; Havedraki, V.;
Rekatas, C. J.; Mai, S. M.; Attwood, D.; Booth, C.; Ryan, A. J.; Hamley, I. W.; Martini, I. 2001,
Macromol. Chem Phys., Vol. 202, pp. 1345-1354.
84. Micellization and Gelation of Triblock Copolymers of Ethylene Oxide and Styrene Oxide in
Aqueous Solution. Yang, Z.; Crothers, M.; Ricardo, N. M. P. S.; Chaibundit, C.; Taboada, P.;
Mosquera, V.; Kelarakis, A.; Havredaki, V.; Mart, L.; Collect, H. J.; Attwood, D.; Heatley, F.;
Booth, C. 2003, Langmuir, Vol. 19, pp. 943-950.
85. Katime, I. Química Física Macromolecular. Universidad del País Vasco : Servicio Editorial del
País Vasco Euskal Herriko , 1994.
86. Bilayers and Interdigitation in Block Copolymer Vesicles. Battaglia, G.; Ryan, A. J. 2005, J.
Am. Chem. Soc., Vol. 127, pp. 8757-8764.
87. Shear-Induced Formation of Multilamellar Vesicles (“Onions”) in Block Copolymers. Zipfel,
J.; Lindner, M.; Tsinou, M.; Alexandridis, P.; Richtering, W. 1999, Langmuir, Vol. 15, pp. 2599-
2602.
88. The evolution of vesicles from bulk lamellar gels. Battaglia, G.; Ryan, A. J. 2005, Nature
Materials, Vol. 4, pp. 869-876.
89. Spontaneous Vesicle Formation in a Block Copolymer System. Bryskhe, K.; Jansson, J.;
Topgaard, D.; Schillén, K.; Olsson, U. J. 2004, J. Phys. Chem. B, Vol. 108, pp. 9710-9719.
90. Association behavior of mixed triblock copoly(oxyalkylene)s (type EBE and ESE) in aqueous
solution. Ricardo, N. M. P. S.; Chaibundit, C.; Yang, Z.; Attwood, D.; Booth, C. 2006, Langmuir,
Vol. 22, pp. 1301-1306.
91. Mixtures of triblock copolymers E62P39E62 and E137S18E137 potential for drug delivery
from in situ gelling micellar formulations. Pinho, M. E. N.; Costa, F. M. L. L.; Filho, F. B. S.;
Ricardo, N. M. P. S.; Yeates, S. G.; Attwood, D.; Booth, C. 2007, Int. J. Pharm., Vol. 328, pp. 95-
98.
92. Creighton, T. E. Proteins: structure and molecular. New York: Freeman, 1993.
93. Nelson, D. L.; Cox, M. M. Principles of biochemistry. New York: Freeman, 2008.
228

94. Are there Pathways for Protein Folding? Levinthal, C. 1968, J. Chim. Phys. Phys. Chim. Biol.,
Vol. 65, pp. 44-45.
95. Protein Folding and Unfolding at Atomic Resolution. Fersht, A. R.; Daggett, V. 2002, Cell ,
Vol. 108, pp. 573-582.
96. Understanding protein folding via free-energy surfaces from theory and experiment.
Dinner, A. R.; Sali, A.; Smith, L. J.; Dobson, C. M.; Karplus, M. 2000, Trends Biochem. Sci. 25
(2000) 331–339., Vol. 25, pp. 331-339.
97. Navigating the Folding Routes. Wolynes, P. G.; Onuchic, J. N; Thirumalai, D. 1995, Science,
Vol. 267, pp. 1619-1620.
98. Folding versus aggregation: Polypeptide conformations on competing pathways. Jahn, T.
R.; Radford, S. E. 2008, Archives of Biochemistry and Biophysics, Vol. 469, pp. 100-117.
99. Towards complete descriptions of the free–energy landscapes of proteins. Vendruscolo, M.;
Dobson, C. M. 2005, Philos. Transact. A Math. Phys. Eng. Sci., Vol. 363, pp. 433-450.
100. Protein Aggregation and Amyloid Fibril Formation by an SH3 Domain Probed by Limited
Proteolysis. Polverino de Laureto, P.; Taddei, N.; Frare, E.; Capanni, C.; Costantini, S.; Zurdo,J.;
Chiti, F.; Dobson, C. M.; Fontana, A. 2003, J. Mol. Biol., Vol. 334, pp. 129-141.
101. Folding funnels, binding funnels, and protein function. Tsai, C. J.; Kumar, S.; Nussinov, R.
1999, Protein Sci., Vol. 8, pp. 1181-1190.
102. Kinetics and thermodynamics of amyloid fibril assembly. Wetzel, R. 2006, Acc. Chem. Res.,
Vol. 39, pp. 671-679.
103. The protofilament structure of insulin amyloid fibrils. Jimenez, J. L.; Nettleton, E. J.;
Bouchard, M.; Robinson, C. V.; Dobson, C. M.; Saibil, H. L. 14, 2002, PNAS, Vol. 99.
104. Hierarchical assembly of β2-microglobulin amyloid in vitro revealed by atomic force
microscopy. Kad, N. M.; Myers, S. L.; Smith, D. P.; Smith, D. A.; Radford, S. E.; Thomson, N. H.
2003, J. Mol. Biol., Vol. 330, pp. 785–797.
105. Einige Bemerkungen über den vegetabilischen Faserstoff und sein Verhältniss zum
Stärkemehl. Schleiden, M. J. 1838, Ann. Physik., Vol. 119, pp. 391-397.
106. On the structural definition of amyloid fibrils and other polypeptide aggregates. Fändrich,
M. 2007, Cell. Mol. Life Sci., Vol. 64, pp. 2066-2078.
107. Amyloids: Not only pathological agents but also ordered nanomaterials. Cherny, I.; Gazit,
E. 2008, Angew. Chem. Int., Vol. 47, pp. 4062-4069.
108. Prediction of the aggregation propensity of proteins from the primary sequence:
Aggregation properties of proteomes. Castillo, V.; Graña-Montes, R.; Sabate, R.; Ventura, S.
2011, Biotechnol. J., Vol. 6, pp. 674-685.
229

109. Unraveling the Mysteries of Protein Folding and Misfolding. Ecroyd, H.; Carver, J. A. 2008,
IUBMB Life, Vol. 60, pp. 769-774.
110. Mechanisms of amyloid fibril formation–focus on domain-swapping. Zerovnik, E.; Stoka,
V.; Mirtic, A.; Guncar, G.; Grdadolnik, J.; Staniforth, R. A.; Turk, D.; Turk, V. 2011, FEBS
Journal, Vol. 278, pp. 2263-2282.
111. Solid-State NMR Study of Amyloid Nanocrystals and Fibrils Formed by the Peptide
GNNQQNY from Yeast Prion Protein Sup35p. van der Wel, P. C. A.; Lewandowski, J. R.; Griffin,
R. G. 2007, J. Am. Chem. Soc., Vol. 129, p. 5117.
112. Structural integrity of β-sheet assembly. Marshall, K. E.; Serpell, L. C. 2009, Biochem. Soc.
Trans., Vol. 37, pp. 671-676.
113. Amyloids Fibrils in Bionanobiotechnology. Waterhouse, S. H.; Gerrad, J. A. 2004, Aust. J.
Chem., Vol. 57, pp. 519-523.
114. Design of model systems for amyloid formation: lessons for prediction and inhibition.
Pastor, M. T.; Esteras-Chopo, A.; de la Paz, M. L. 2005, Current Opinion in Structural Biology,
Vol. 15, pp. 57-63.
115. A designed system for assessing how sequence affects to conformational transitions in
proteins. Ciani, B.; Hutchinson, E. G.; Sessions, R. B.; Woolfson, D. N. 2002, J Biol Chem, Vol.
277, pp. 10150-10155.
116. Mutational analysis of designed peptides that undergo structural transition from -helix
to -sheet and amyloid fibril formation. Takahashi, Y.; Ueno, A.; Mihara, H. 2000, Structure
Fold Des., Vol. 8, pp. 915-925.
117. Amyloid architecture: complementary assembly of heterogeneous combinations of three
or four peptides into amyloid fibrils. Takahashi, Y.; Ueno, A.; Mihara, H. 2002, Chem. Bio.
Chem., Vol. 3, pp. 637-642.
118. Exploring amyloid formation by a de novo design. Kammerer, R. A.; Kostrewa, D.; Zurdo,
J.; Detken, A.; Garcia-Echeverria, C.; Green, J. D.; Muller, S. A.; Meier, B. H.; Winkler, F. K.;
Dobson, C. M. 2004, Proc. Natl. Acad. Sci., Vol. 101, pp. 4435-4440.
119. Hacking the code of amyloid formation. Pastor, M. T.; Esteras-Chopo, A.; Serrano, L.
2007, Prion, Vol. 1, pp. 1-14.
120. Why are “natively unfolded” proteins unstructured under phisiologic condition? Uversky,
V. N.; Gillespie, J. R.; Fink, A. L. 2000, PROTEINS: Structure, Function, and Genetics, Vol. 41, pp.
415-427.
121. Conformational constraints for amyloid fibrillation: the importance of being unfolded.
Uversky, V. N.; Fink, A. L. 2004, Biochim. Biophys. Acta, Vol. 1698, pp. 131-153.
122. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.
Colon, W.; Kelly, J. W. 1992, Biochemistry, Vol. 31, pp. 8654-8660.
230

123. Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Chiti, F.;
Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, G. M. 1999, Proc. Natl.
Acad. Sci., Vol. 96, pp. 3590-3594.
124. Partially Unfolded States of β2-Microglobulin and Amyloid Formation in Vitro. McParland,
V. J.; Kad, N. M.; Kalverda, A. P; Brown, A.; Kirwin-Jones, P.; Hunter, M. G.; Sunde, M.;
Radford, E. S. 2000, Biochemistry, Vol. 32, pp. 8735-8746.
125. Assembly of Amyloid Protofibrils via Critical Oligomers-A Novel Pathway of Amyloid
Formation. Modler, A. J.; Gast, K.; Lutsch, G.; Damaschun, G. 2003, J. Mol. Biol., Vol. 325, pp.
135-148.
126. Competing Pathways Determine Fibril Morphology in the Self-assembly of β2-
Microglobulin into Amyloid. Gosal, W. S.; Morten, I. J.; Hewitt, E. W.; Smith, D. A.; Thomson,
N. H.; Radford, S. E. 2005, J. Mol. Biol., Vol. 351, pp. 850-864.
127. Direct Observation of Oligomeric Species formed in the Early Stages of Amyloid Fibril
Formation using Electrospray Ionisation Mass Spectrometry. Smith, A. M.; Jahn, T. R.;
Ashcroft, A. E.; Radford, S. E. 2006, J. Mol. Biol., Vol. 364, pp. 9-19.
128. Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways.
Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Voller, S. S.; Benedek, G. B.; Teplow, D. B. 2003,
Proc. Natl. Acad. Sci., Vol. 100, pp. 330-335.
129. Small Molecule Inhibitors of Aggregation Indicate That Amyloid β Oligomerization and
Fibrillization Pathways Are Independent and Distinct. Necula, M.; Kayed, R.; Milton, S.; Glabe,
C. G. 2007, J. Biol. Chem., Vol. 282, pp. 10311-10324.
130. Thermodynamic features of the thermal unfolding of human serum albumin. Picó, G. A.
1997, International Journal of Biological Macromolecules, Vol. 20, pp. 63-73.
131. Atomic structure and chemistry of human serum albumin. He, X. M.; Carter, D. C. 1992,
Vol. 358, pp. 209-215.
132. Two-dimensional/attenuated total reflection infrared correlation spectroscopy studies on
secondary structural changes in human serum albumin in aqueous solutions: pH-dependent
structural changes in the secondary structures and in the hydrogen bondings of. Murayama, K.;
Wu, Y.; Czarnik-Matusewicz, B.; Ozaki, Y. 2001, J. Phys. Chem. B, Vol. 105, pp. 4763-4769.
133. Spectroscopic studies on the effect of temperature on pH-induced folded states of human
serum albumin. Shaw, A. K.; Pal, S. K. 2008, Journal of Photochemistry and Photobiology B:
Biology, Vol. 90, pp. 69-77.
134. Unfolding of acrylodan-labeled human serum albumin probed by steady-state and timeresolved
fluorescence methods. Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Bright, F.
V. 1998, Biophysical Journal, Vol. 75, pp. 1084–1096.
135. Thermal stability of bovine serum albumin DSC study. Michnik, A. 2003, Journal of
Thermal Analysis and Calorimetry, Vol. 71, pp. 509-519.
231

136. Dilatational Rheology of BSA Conformers at the Air/Water Interface. Cascão Pereira, L. G.;
Theódoly, O.; Blanch, H. W.; Radke, C. J. 2003, Langmuir, Vol. 19, pp. 2349-2356.
137. The three recombinant domains of human serum albumin: structural characterization and
ligand binding properties. Dockal, M.; Carter, D. C.; Rüker, F. 41, 1999, The journal of
biological chemistry, Vol. 274, pp. 29303–29310.
138. The behavior of polyamino acids reveals an inverse side chain effect in amyloid structure
formation. Fändrich, M.; Dobson, C. M. 2002, The EMBO J., Vol. 21, pp. 5682–5690.
139. Existence of different structural intermediates on the fibrillation pathway of human serum
albumin. Juárez, J.; Taboada, P.; Mosquera, V. 2009, Biophysical Journal, Vol. 96, pp. 2353-
2370.
140. Influence of electrostatic interactions on the fibrillation process of human serum albumin.
Juárez, J.; Taboada, P.; Mosquera, V. 2009, J. Phys. Chem. B, Vol. 113, pp. 10521-10529.
141. Additional supra-self-assembly of human serum albumin under amyloid-like-forming
solution conditions. Juárez, J.; Taboada, P.; Goy-López, S.; Cambón, Adriana; Madec, M. B.;
Yeates, S. G.; Mosquera, V. 2009, J. Phys. Chem. B, Vol. 113, pp. 12391-12399.
142. Electrochemical devices made from conducting nanowire networks self-assembled from
amyloid fibrils and alkoxysulfonate PEDOT. Hamedi, M.; Herland, A.; Karlsson, R. H.; Inganäs,
O. 2008, Nano Lett., Vol. 8, pp. 1736-1740.
143. Nanomaterials: amyloids reflect their brighter side. Mankar, S.; Anoop, A.; Sen, S.; Maji,
S. K. 2011, Nano Reviews, Vol. 2, pp. 1-12.
144. Nanomechanics of functional and pathological amiloid materials. Knowles, T. P. J.;
Buehler, M. J. 2011, Nature nanotechnology, Vol. 6, pp. 469-479.
145. Seeding-induced self-assembling protein nanowires dramatically increase the sensitivity of
immunoassays. Men, D.; Guo, Y. C.; Zhang, Z. P.; Wei, H. P.; Zohug, Y. F.; Cui, Z. Q.; Liang, X.
S.; Li, K.; Leng, Y.; You, X. Y.; Zhang, X. E. 2008, Nano Lett., Vol. 9, pp. 2246-2250.
146. Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly:
implications for amyloid fibril formation and materials science. Lashuel, H. A.; LaBrenz, S. R.;
Woo, L.; Serpell, L. C.; Kelly, J. W. 2000, J. Am. Chem. Soc., Vol. 12, pp. 5262-5277.
147. Ultrathin silver nanowires produced by amyloid biotemplating. Malisauskas, M.; Meskys,
R.; Morozova-Roche, L. A. 2008, Biotechnol. Prog., Vol. 2004, pp. 1166-1170.
148. LSPR-based nanobiosensors. Sepúlveda, B.; Angelomé, P. C.; Lechuga, L. M.; Liz-Marzán,
L. M. 2009, Nano today, Vol. 4, pp. 244-251.
149. Shape-controlled synthesis of silver nanoparticles for plasmonic and sensing applications.
Cobley, C. M.; Skrabalak, S. E.; Xia, Y. 2009, Plasmonic, Vol. 4, pp. 171-191.
150. A comparison study of the catalytic properties of Au-based nanocage, nanoboxes, and
nanoparticles. Zeng, J.; Zhang, Q.; Chen, J.; Xia, Y. 2010, Nano lett., Vol. 10, pp. 30-35.
232

151. SERS-based diagnosis and biodetection. Alvarez-Puebla, R. A.; Liz-Marzán, L. M. 2010,
Small, Vol. 6, pp. 604-610.
152. A refined solution structure of hen lysozyme determined using residual dipolar coupling
data. Schwalbe, H.; Grimshaw, S. B.; Spencer, A.; Buck, M.; Boyd, J.; Dobson, C. M.; Redfield,
C.; Smith, L. J. 2001, Protein Science, Vol. 10, pp. 677-688.
153. Amyloid fibril formation of hen lysozyme depends on the instability of the C-helix (88-99).
Harada, A.; Azakami, H.; Kato, A. 2008, Biosci. Biotechnol. Biochem., Vol. 72, pp. 1523-1530.
154. Comparative study of inactivation and conformational change of lysozyme induced by
pulsed electric fields and heat. Zhao, W.; Yang, R. 2008, Eur. Food. Res. Technol., Vol. 228, pp.
47-54.
155. Thermally induced fibrillar aggregation of hen egg white lysozyme. Arnaudov, L. N.; de
Vries, R. 2005, Biophysical Journal, Vol. 88, pp. 515-526.
156. Catalytic properties of gold nanoparticles immobilized on the surfaces of Nanocarriers.
Chen, X.; Zhao, D.; An, Y.; Shi, L.; Hou,W.; Chen, L. 2010, J. Nanopart. Res., Vol. 12, pp. 1877-
1887.
157. Preparation of gold-dendrimer nanocomposites by laser irradiation and their catalytic
reduction of 4-nitrophenol, immobilization and Recovery of Au Nanoparticles from anion
exchange resin. Hayakawa, K.; Yoshimura, T.; Esumi, K. 2003, Langmuir, Vol. 19, pp. 5517-
5521.
158. A nanoreactor framework of a Au@SiO2 yolk/shell structure for catalytic reduction of pnitrophenol.
Lee, J.; Park, L. C.; Song, H. 2008, Adv. Mater. 2008, 20, 1523–1528., Vol. 20, pp.
1523-1528.
159. Responsive catalysis of thermoresponsive micelle-supported gold nanoparticles. Wang, Y.;
Wei, G.; Zhang, W.; Jiang, X.; Zheng, P.; Shi, L.; Dong, A. 2007, J. Mol. Catal. A: Chem., Vol.
266, pp. 233-238.
160. Magnetic nanoparticle assemblies on denatured DNA show unusual magnetic relaxivity
and potential applications for MRI. Byrne, S. J.; Corr, S. A.; Gun'ko, Y. K.; Kelly, J. M.;
Brougham, D. F.; Ghosh , S. 2004, Chem. Commun., pp. 2560-2561.
161. Linear assemblies of magnetic nanoparticles as MRI contrast agents. Corr, S. A.; Byrne, S.
J.; Tekoriute, R.; Meledandri, C. J.; Bougham, D. F.; Lynch, M.; Kerskens, C.; O'Dwyer, L.;
Gun'ko, Y. K. 2008, J. Am. Chem. Soc., Vol. 130, pp. 4214-4215.
162. Wrap–bake–peel process for nanostructural transformation from bold beta-FeOOH
nanorods to biocompatible iron oxide nanocapsules. Piao, Y.; Kim, J.; Na, H. B.; Kim, D.; Baek,
J. S.; Ko, M. K.; Lee, J. H.; Shokouhimehr, M.; Hyeon, T. 2008, Nat. Matter., Vol. 7, pp. 242-
247.
233